Treatment of bacterial infections

ABSTRACT

An assay for compounds useful in the treatment of a bacterial induced coagulation disorder has the following steps: 
     a) incubating a plasma sample with a strain of bacteria; 
     b) adding a compound to be assayed to the plasma sample before, during or after step (a); 
     c) conducting an activated partial thromboplastin time test; 
     d) determining the clotting time.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/194,098, filed Jun. 25, 1999; which application is a 371conversion of PCT Application No. PCT/SE97/00825, filed May 20, 1997.

TECHNICAL FIELD OF THE INVENTION

The invention relates to assays for the detection of compounds usefulfor the treatment of the bacterial infection and in particularconditions associated with infection by curli-expressing gram negativebacteria and streptococci.

BACKGROUND OF THE INVENTION

Some micro-organisms such as Streptococci, Salmonella, E.coli andS.aureus may cause severe invasive infections such as sepsis or septicshock. Fever, hypotension and bleeding disorders are common symptoms ofsepsis and septic shock. Invasive infections caused by these bacteriamay result from resistance to antibiotics or defects in the immunesystem of the infected individual.

The endogenous or intrinsic pathway of inflammation and coagulation istriggered by assembly of the contact phases system. The contact phasesystem is orchestrated by three serine proteases, factor XI (hereinafterreferred to as F XI), factor XII (F XII) and plasma kallikrein (PK) aswell as the non-enzymatic co-factor H-kininogen (HK). H-kininogen formsequimolar complexes with F XI and PK. The local activation of thisproteolytic system triggers different cascades such as thesurface-dependent activation of blood coagulation, fibrinolysis, kiningeneration and inflammation reactions.

Events which allow the assembly of the contact phase components lead toconversion of F XII to the active enzyme F XIIa which triggers thecontact phase system. Partially activated V XII cleaves PK which isbound to surfaces via HK. By a mechanism of reciprocal activation, PKamplifies the activity of F XII and in addition PK cleaves HK to releasethe nona peptide bradykinin. Activated F XII cleaves F XI into itsactive form which leads to initiation of the intrinsic pathway ofcoagulation.

Bradykinin and the physiologically important related peptides kallidin(Lys-bradykinin) and Met-Lys bradykinin, contract smooth muscle forexample to produce diarrheoa and inflammatory bowel disease and asthma,lower blood pressure, mediate inflammation as in allergies, arthritisand asthma, participate in blood clotting and complement-mediatedreaction in the body, mediate rhinitis (viral, allergic andnon-allergic) and are over produced in pathological conditions such asacute pancreatitis, hereditary angioneurotic edema, post-gastrectomydumping syndrome, carcinoid syndrome, anaphylactic shock, reduced spermmobility and certain other conditions.

As a result of the fact that bradykinin is involved in all the abovementioned clinical indications, a large number of bradykinin antagonistshave been developed. Such antagonists are disclosed in e.g. U.S. Pat.No. 4,693,993. Wirth et al., Can. J. Physiol. Pharmacol. Vol. 73 pp797-804 presents clinical studies regarding administering bradykininantagonists for treating post-operative pain, asthma, anaphylactoidreactions, systemic inflammatory response syndrome, and suspectedsepsis, head injury and hantavirus infections. A review on clinicalapplications of bradykinin antagonists can be found in Cheronis et al.eds: Proteases, Protease Inhibitors and Protease Derived Peptides pp167-176. Although these citations disclose administration of bradykininantagonists for treating sepsis, it is evident that the suspected sepsistreated by said antagonists it not primarily caused by bacterialinfections. Accordingly the citations are completely silent about usingbradykinin antagonists for treating bacterial infections. Moreover it isevident that bradykinin antagonists have been administered to relievethe symptoms of inflammation.

SUMMARY OF THE INVENTION

Some of the symptoms of sepsis may be explained by activation of thecontact phase system. However the precise mechanisms involved are notwell understood. The inventors have now established the initial stagesinvolved in activation of the contact phase system during bacterialinfection. These studies have led to new assays being proposed toidentify agents which specifically target the reactions resulting frombacterial activation of the contact phase system. These studies alsodemonstrate the disruption of the normal coagulation pathway inbacterial infections and thus propose new substances to treat conditionssuch as sepsis and septic shock.

The invention provides a number of assays to identify useful agents forthe treatment of bacterial infections. In a first aspect, the inventionprovides an assay for compounds useful in the treatment of abacterial-induced coagulation disorder, which assay comprises the stepsof:

a) incubating a plasma sample with a strain of bacteria;

b) adding a compound to be assayed to the plasma sample before, duringor after step a);

c) conducting an activated partial thromboplastin time aPTT test on thesample after steps a) and b);

d) determining the clotting time.

In a second aspect, the invention provides an assay for compounds usefulin the treatment of a bacterial-induced coagulation or inflammatorydisorder, which assay comprises the steps of:

a) incubating a strain of bacteria with one or more contact phaseproteins selected from the group consisting of H-kininogen,prekallikrein, factor XII and factor XI;

b) adding a compound to be assay before, during or after step a); and

c) determining the binding of the contact phase proteins to thebacterial surface.

In a further aspect, the invention provides an assay for compoundsuseful in the treatment of a bacterial induced coagulation orinflammatory disorder, which assay comprises the steps of:

a) incubating a strain of bacteria with one or more contact phaseproteins selected from the group consisting of H-kininogen,prekallikrein, factor XII and factor XI

b) adding the compound to be assayed before, during or after step a);and

c) determining the activation of the or each contact phase protein.

By monitoring the interactions between bacteria and the contact phasecomponents, agents can be identified which may be useful in thetreatment of shock, sepsis and bleeding disorders seen in more severebacterial infections.

DESCRIPTION OF THE FIGURES

In the figures:

FIG. 1 generally outlines a major route for the formation of bradykinin.Factor XII zymogen is activated either by a part of the clottingcascade, negatively charged surfaces or (disclosed herein) bacterialsurface proteins. Activated factor XII then activates prekallikreinthereby forming kallikrein, which in turn cleaves kininogens in order toform bradykinin. Bradykinin has a short half-life and is quicklyinactivated by kininase.

FIG. 2 discloses the structure of H-kininogen and assembly of contactfactors. Part A: HK comprises six domains: D 1 to D3 have cystatin-likestructure and represent potent cysteine proteinase inhibitors (D2, D3)and expose a cell-binding site (D3). Domain D4 bears the kinin segment.D5_(H) exposes another cell-binding site, and D6_(H) holds thezymogen-binding site for prekallikrein and factor XI (shaded). Stepwiseproteolysis of HK by α-kallikrein at site 1 (marked by an arrowhead)releases a 63 kDa heavy chain with the bradykinin segment stillattached, and a 58 kDa light chain; the two chains are interconnected bya single disulphide bridge. Bradykinin is released following cleaveageat site 2. Secondary cleaveage at site 3 converts the 58 kDa light chaininto the 45 kDa light chain fragment. The epitopes of antibodies α-BKand HLK 16 are indicated below the chains. Part B: HK binds tonegatively charged surfaces and brings plasma prekallikrein (PK) andfactor X(FXI) in proximity to surface-bound factor XII (FXII).Reciprocal proteolytic activation of FXII and PK converts theseproenzymes to active proteinase (Henderson et al. 1994, Blood vol. 84,pp 474-482). Activated FXII activates FXI which propagates the intrinsiccoagulation pathway via factor IX, whereas activated PK (kallikrein)cleaves HK and releases bradykinin. The triggering event (marked by anasterisk) that starts the contact phase activation is still ill-defined.

FIG. 3 discloses the gross structure of mammalian kininogens.H-kininogen and L-kininogen share their heavy chain domains, D 1 to D4,and differ in their light chain domains, D5_(H)/D6_(H) and D5_(L),respectively. Domains D I to D3 are of cystatin-like structure; domainD2 inhibits calpain and papain-like cystein proteinases whereas D3inhibits only papain-like enzymes and exposes a cell-binding site. Thekinin segment is located in domain D4. Domain D5_(H) of H-kininogenexposes a high-affinity cell-binding site which is also used bystreptococcal M protein. Domain D6_(H) contains the overlapping bindingsites for prekallikreinand factor XI. The function of D5_(L) ofL-kininogen is unknown. Protein-sensitive regions flanking the kininsegment are indicated by pairs of solid arrowheads.

FIG. 4 shows analysis of the interactions between H-kininogen and MIprotein, and H kininogen and plasma prekallikrein. This is illustratedby an overlay plot of the binding of M 1 protein (A) and plasmaprekallikrein (B) to immobilized HK using plasmon resonancespectroscopy. Increasing concentrations of MI protein (12.5, 25, 50, and100 μg/ml) or plasma prekallikrein (3.13, 6.25, 12.5 and 25 μg/ml) wereapplied for 3 min each during the association phase. Dissociation ofbound proteins was measured following injection of buffer alone.

FIG. 5, which is of the same type as FIG. 4, shows that M 1 protein andplasma prekallikrein do not compete for the same binding-site inH-kininogen. M1 protein (30 μl, 50 μg/ml) and plasma prekallikiein (30μl, 10 μg/ml) were applied to a sensor chip coupled with HK.Furthermore, a 30 μl sample containing both proteins (50 μg/ml M proteinand 10 μg/ml plasma prekallikrein) were assayed. Samples were alsoapplied as above except that plasma kallikrein was added followingcomplex formation between M1 protein and HK.

FIG. 6 discloses a Western blot analysis revealing that H-kininogenbound to the streptococcal surface is cleaved. Following incubation withhuman plasma, bacteria of strains AP 1, AP46, and the M protein-negativemutant strain AP74 were treated with glycine buffer, pH 2.0, tosolubilize plasma proteins bound to the bacteria. The resultingsupernatants were subjected to SDS-PAGE (10%) under reducing conditions.One gel with the sample was run. It was electroblotted to a PVDFmembrane. The membrane was probed with monoclonal antibodies against thelight COOH-terminal chain of HK, followed by peroxidase-labeledsecondary antibodies (BLOT).

FIG. 7 relates to binding of radiolabeled H-kininogen to variousbacterial species. In total 118 strains, all isolated from patients withsepsis, belonging to 18 different bacterial species were tested forbinding of [¹²⁵I]-HK. Each dot represents one strain and figures withinparenthesis indicate the number of tested strains of a given species.

FIG. 8 relates to absorption of H-kininogen from human plasma bydifferent strains of E. coli. Bacteria were incubated with human plasmafor 60 min at 37° C. Following extensive washing proteins bound to thebacteria were eluted at pH 2.0. The eluted proteins were subjected toSDS-PAGE (10%) run under reducing conditions and stained with CoomassieBlue (STAIN). A replica of the gel was electrotransferred to a PVDFmembrane which was probed with monoclonal antibody HKL 16 followed bysecondary peroxidase-labeled antibody (BLOT).

FIG. 9 shows binding of radiolabeled H-kininogen to curli-expressing E.coli. A constant amount of [¹²⁵I]-labeled HK (10⁴ cpm corresponding to 1ng in 225 μl) was incubated with different numbers of curliated Ymel (•)or non-curliated Ymel-1 (°)bacteria for 60 min at 37° C. Binding isexpressed as the percentage of radioactivity present in the bacterialpellet in relation to the total radioactivity applied per tube (leftpart of the figure). Increasing numbers of Ymel bacterial were incubatedwith 1 ml fresh human plasma. Non-curliated Ymel-1 bacteria were addedto give 5×10¹¹ bacteria throughout the tests. Following incubation,proteins bound to the cells were eluted and separated by SDS-PAGE (10%)under reducing conditions, and electrotransferred to a PVDF membrane.The membrane was probed with antibody HKL 16 followed by secondaryperoxidase-labeled antibodies (right part of the figure).

FIG. 10 discloses that H-kininogen binds to purified bacterial proteinsubunits in Western blots. Curll subunits purified from Ymel bacteria(A), streptococcal M1 protein (B), and a COOH-terminal fragment thereof,S-C3 (C), were separated by SDS-PAGE (13.6%) under reducing conditionsand strained with Coomassie Blue (STRAIN). Proteins were blotted ontoPVD17 membranes and probed with [¹²⁵I]-HK (BLOT).

FIG. 11 relates to a Scatchard plot for the binding of H-kininogen topolymeric curli. Constant amounts of polymeric curli and [¹²⁵I]-HK wereincubated with varying amounts of unlabeled HK. Free HK was separatedfrom HK bound to curli by centrifugation. The radioactivity of theresulting pellet was measured, and calculation of the affinity constantwas done according to Scatchard (1949), The attractions of proteins forsmall molecule and ions, Ann. N. Y. Acad. Sci. vol. 5 1: pp. 660-672.

FIG. 12 describes binding of L-kininogen and contact factors tocurliated E.coli. 2×10⁹ curli-expressing Ymel (□) or curli-deficientYmel-1 (□) bacteria were incubated separately with 0.5-1 ng (10⁴ cpm)H-kininogen (HK), L-kininogen (LK), prekallikrein (PK), factor XI (FXI),or factor XII (FXH); all proteins were from human plasma. Incubation for60 min at 37° C. was followed by washing, centrifugation, and measuringof the radioactivity of the pellet. Binding was expressed as percentageof the total amount of [¹²⁵I]-labeled protein added to the bacteria.Mean values±SEM are indicated.

FIG. 13 shows that HK absorbed by and eluted from curliated E. coli ispartially cleaved. Following incubation with human fresh plasma,proteins were eluted from curliated Ymel (A) or curli-deficient Ymel-1(B) bacteria with pH 2.0, and subjected to SDS-PAGE (10%) under reducingor non-reducing conditions followed by staining with Coomassie Blue(STAIN). Replicas of these gels were probed with antibodies α-BK or HLK16, followed by peroxidase-labeled secondary antibodies (BLOT).

FIG. 14 discloses that plasma killikrein cleaves surface-boundH-kininogen and releases bradykinin. Curliated Ymel bacteria werepreincubated with human plasma, washed, and incubated with buffer alonefor 40 min (A) or with activated plasma kallikrein (0. 1 mg/ml) for 5min (B), 10 min (C), 20 min (D), and 40 min (E). Proteins bound to thebacteria were eluted with pH 2.0 and subjected to SDS-PAGE (10%),electroblotted onto a PVDF filter and probed with plyclonal antibodiesagainst bradykinin (α-BK), followed by secondary peroxidase-labeledantibodies.

FIG. 15 relates to cleavage of H-kininogen by the streptococcal cysteineproteinase (SCP). H-kininogen (30 μg) was incubated with 0.07 μg of SCP(molar ratio of 100:1). After 15 min (lane 2), 30 min (3), 60 min (4),120 min (5), or 180 min (6) of incubation aliquots of the reactionmixture (4 μg protein each) were separated by SDS-PAGE (10% v/v) underreducing conditions, followed by staining with Coomassie Brilliant Blue.For control H-kininogen incubated for 180 min in the absence of SCP wasapplied (lane 1). Note that a small amount of the purified H-kininogenexists in its kinin-free two chain form. Standard molecular markerproteins were run simultaneously (not shown); their relative positionsare indicated on the left.

FIG. 16 shows an immunoprint analysis of H-kininogen cleavage products.Aliquots from the reaction mixture of H-kininogen (30 μg) and SCP (0.07μg) were removed after 15 min (lane 2), 30 min (3), 60 min (4), 120 min(5), or 180 min (6) and separated by SDS-PAGE followed byelectrotransfer onto nitrocellulose. For control native H-kininogenincubated for 180 min in the absence of SCP (1) was applied. Blots wereincubated with HKH 15 antibody (A), α-BK antibodies (B), or HKL 9antibody (C). Bound antibodies were visualized with peroxidase labelledanti-mouse or anti-rabbit immunoglobulins and the chemiluminescencetechnqiue. The relative locations of the antibodies' target epitopes,are indicated on the bottom; the domain designation is that of FIG. 3.Note that α-BK shows a high affinity for kininogen fragments rather thanfor the uncleaved H-kininogen.

FIG. 17 discloses that Ca²⁺ release from intracellular stores induced byH-kininogen cleavage products. Confluent human fibroblasts loaded withfura-2 were incubated with untreated H-kininogen (A), and H-kininogencleaved by SCP for 30 min (B), 60 min (C), or 120 min (D) at 37° C. Theintracellular Ca²⁺ release was measured as the ratio of fluorescence atexcitation wavelengths of 340 nm and 380 nm, respectively.

FIG. 18 relates to H-kininogen cleavage in plasma. Human plasma (100 μl)was incubated with 3.2 μg of SCP. Samples were taken after 15 min (lane2), 30 min (3), 45 min (4), 60 min (5), or 90 min (6) of incubation andseparated by SDS-PAGE followed by the transfer of the proteins ontonitrocellulose and immunostaining by antibodies against nativeH-kininogen (AS88; panel A) or to BK (α-BK; B). For control, plasma wasincubated in the absence of SCP for 90 min (1). The relative positionsof the human plasma kininogens are marked on the right.

FIG. 19 shows the time course of prekallikrein activation by variousproteinases. Plasma prekallikrein was incubated for 1 h with factor XIIin a molar ratio of 100:1 (□) or with SCP in molar ratio of 100:1 (⋄);and 10:1 (∘). At the indicated time points aliquots of the reactionmixtures were removed, and their amidolytic activity tested by achromogenic substrate assay (H-D-Pro-Phe-Arg-pNA). For control,prekallikrein incubated in the absence of SCP (Δ), or SCP alone (∇) weretested.

FIG. 20 discloses kinin generation by SCP in plasma followed by ELISA.Human plasma (100 μl) was incubated with 3.2 μg of SCP (▪), aliquots ofthe reaction mixture were removed at the time points indicated, andassayed for their kinin concentration by a competitive ELISA. Forcontrol plasma was incubated under identical conditions except that SCPwas omitted (▪). Note that the lower detection limit for bradykinin isapproximately 10⁷ mol/l of plasma.

FIG. 21 shows cleavage of plasma kininogens by SCP in vivo. Panel A:Mice were injected i.p. with 0.5 mg of purified SCP. Plasma samples weredrawn from the animals after 60 min (lane 2), 150 min (lane 3), and 300min (lane 4). Alternatively, 0.5 mg SCP mixed with 0.2 mgZ-Leu-Val-Gly-CHN₂ was injected, and a plasma sample from this mouse wastaken 300 min after injection (lane 5). For control, plasma from a mouseinjected with PBS alone was used (lane 1). One 82 of each sample wasseparated by SDS-PAGE, transferred to nitrocellulose and immunostainedwith antibodies to bradykinin (α-BK). The relative positions of themarket proteins are given to the left, and those of the mouse plasmakininogens are indicated to the right. Panel B: Mice were injected(i.p.) with PBS alone (lane 1), 0. 1 mg of SCP (lane 2), 0.2 mg of SCP(lane 3), 0.3 mg of SCP (lane 4), 0.4 mg of SCP (lane 5), or 0.5 mg ofSCP (lane 6). Plasma samples were taken 300 min after injection.

FIG. 22 relates to cleavage of plasma kininogens by S. pyogenes in vivo.Mice were injected i.p. with 0.5 mg of purified SCP or with living S.pyogenes bacteria (3×10⁸ cells) diluted in 0.5 ml PBS. A. Plasma samples(1 μl each) from animals injected with PBS alone (lane 1), with purifiedSCP (lane 2), or with S. pyogenes bacteria (lane 3) were run on SDS-PAGEand stained with Coomasie Brilliant Blue. B. An identical replica onnitrocellulose was probed with antibodies to bradykinin (α-BK). Standardmolecular marker proteins were run simultaneously (not shown); theirrelative positions are indicated on the left. The positions of mousekininogens are marked.

FIG. 23. Binding of radiolabelled contact phase proteins to curliatedE.coli and S.typhimurium. [ ¹²⁵I]labelled F XI, F XII, PK or HK wereincubated with suspensions of the bacterial strains YMel, YMel-1 fromE.coli or SRIIB, CsgA, CsgB from S.typhimurium. Binding is expressed asthe percentage of bound vs. total radioactivity. Data representsmeans±S.D. of 3 experiments each done in duplicate

FIG. 24. A. Limited proteolysis of contact phase factor XII followingincubation with S.typhimurium and E.coli. [ ¹²⁵I]labelled F XII (lane 1)was incubated with the bacterial strains SRIIB or YMel for 10 min (2),15 mins (3), 30 min (4), and 45 min (5). Samples were run on SDS-PAGEunder reducing conditions and visualized by autoradiography B and C.Activation of contact phase factors by S.typhimurium and E.coli. In B,[¹²⁵I]-PK was incubated for 10 min with SRIIB or YMel in the absence (−)or presence (+) of unabeled F XI, F XII and HK and centrifuged. Thepellets were dissolved and run on SDS-PAGE under reducing conditionsfollowed by autoradiography. In C, same as in B except that [¹²⁵I]-HKwas used, and unlabeled PK but not HK was present in the incubationbuffer.

FIG. 25. A. Generation of bradykinin at the bacterial surface. Humanplasma was incubated with strains YMel, YMel-1, SRIIB, CsgA or CsgB. Theamount of bradykinin (BK; pg/10¹⁰ cells) present in the incubationmixtures was measured by a competitive radioimmunoassay. For controlnormal plasma (a) and plasma incubated in the absence of bacteria (b)were incubated. B. Effects of bacteria on the activated partialthromboplastin time (aPTT). Normal human plasma was pre-incubated for 30sec with strains YMel, YMel-1, SRIIB, CsgA and CsgB. The clotting wasinitiated by adding kaoline and CaCl₂. For control plasma was incubatedwith vehicle alone. The dose dependent effect of YMel on aPTT wasstudies with increasing number of bacteria. Data represent means=S.D. oftwo experiments each done in duplicate.

FIG. 26. A. In vivo effect of curliated E.coli on the clotting time.Three mice in each group were intravenously injected with YMel or YMel-1bacteria in PBS. Blood was drawn one hour after infection and the aPTTof the resulting plasma samples were determined. Control samples werefrom the mice infected with PBS alone. B. Depletion of plasma fibrogenby curliated bacteria. Plasma samples generated as described above, werediluted {fraction (1/10)}, {fraction (1/20)}, {fraction (1/40)} etc, in0.15 M NaCl. Activated thrombin was added, and after 15 min the maximumdilution allowing fibrin polymerization was determined.

FIG. 27. Activation of contact phase system on the surface of AP1 andAP6. ¹²⁵I-HK 25 μl of a solution containing radi0-labeled HK (4.6×10⁻¹²M) was incubated with 0.2 ml AP1 and AP6 (2×10⁸ cells/ml) in the absenceor the presence of non-labeled F XII (3.0×10⁻¹² M), PK (3.9×10⁻¹² M),and F XI (0.2×10⁻¹² M F XI) marked by plus or minus. After 10 minsamples were centrifuged and run on SDS-PAGE under reducing conditionsfollowed by autoradiography. ¹²⁵I-PK Radio-labeled plasmakallikrein wasincubated in the presence and the absence of non-labeled F XII, HK and FXI with AP1 and AP6 using the same concentrations as described above.

FIG. 28. Proteolytic activity on the surfaces of AP1 and AP6 aftercontact with plasma. 0.5 ml of human plasma was incubated with 0.5 ml ofthe strains AP1, AP6 and CsgA (2×10¹⁰ cells/ml) for 10 mins at roomtemperature. Bacteria were washed three times and amidolytic activitieswere tested by chromogenic substrate assays for F XI (S-2366) and for FXII/PK (S-2302). As a control, the strain CsgA was used, which fails tobind any of the contact phase factors.

FIG. 29. Effects of bacteria on the partial thromboplastin time (aPTT).

Panel A—30 μl of a bacterial solution of the strains AP1, AP6 and CsgA(2×10¹⁰ cells/ml) were incubated with 100 μl of sodium citrate treatedplasma for 30 s. The clot formation was initiated by adding kaolinreagent and CaCl₂, and subsequently the partial thromboplastin time(aPTT, in seconds) was measured. As a control plasma incubated with onlybuffer was measured.

Panel B—Bacteria and plasma were pre-incubated as described above. Butthe clot formation was initiated in the absence of kaolin. As a controlplasma incubated with only buffer was used.

Panel C—0.5 ml sodium citrate treated plasma were incubated with 0.5 mlof the strains AP1, AP6 and CsgA (2×10¹⁰ cells/ml) dissolved in the samebuffer and incubated for 30 min at room temperature. After the bacteriawere removed by centrifugation, 100 μl of the resulting solution wereused to measure the aPTT. As a control, only buffer was added to plasma.

DETAILED DESCRIPTION OF THE INVENTION

We have demonstrated that the components of the contact phase systembind to the surface of some bacteria. In particular H kininogen is shownto bind to the surface of Streptococci and in particular to S. pyogenes.HK also binds to the surface of curli-expressing bacteria such ascertain strains of E. coli and Salmonella. HK is also shown to releasebradykinin when in contact with Staphylococcus aureus. Bradykinin isreleased from the bacterial bound HK in the presence of, for example,plasma killikrein.

Release of bradykinin from the bacterial surface stimulates aninflammatory response. Bradykinin is also a potent vasoactive peptide.

The other contact phase proteins are also shown to bind to the bacterialsurface. The binding of prekallikrein and factor XI is mediated throughthe binding of HK. Factor XII also binds to the bacterial surface.Activated factor XII plays a role in the inflammatory cascade andcoagulation cascade. In particular we have not found that the presenceof bacteria which bind to the contact phase proteins and in particularwhich bind to F XII increases the clotting time of a plasma sample.

This has a further implications on the ways to treat severe bacterialinfection and proposes the use of coagulation agents which enhance theability of the plasma to form clots. Given that the bacteria appear tobenefit from the binding of the contact phase components and use theinflammatory or coagulation cascades to assist in spread and virulence,the interactions between bacteria and the contact phase proteins can beused to assay for agents which interfere or modify these interactions.Monitoring the effect of HK and F XII binding to bacteria can also beused to identify new anti-inflammatory agents or coagulation agentswhich may be used in general to treat bleeding disorders andinflammation which are not necessarily caused by bacterial infection.

Candidate substances

A substance which interferes with activation of the contact phase systemat the bacterial surface may do so in several ways. It may directlyinterfere with the binding of one or more of the contact phasecomponents to the bacterial surface. Alternatively, it may interferewith the interaction of the contact phase components once bound on thebacterial surface or prevent activation of the contact phase system.

Candidate substances of each of these types may conveniently be screenedby in vitro binding assays, for example, as described below. They mayalso be screened by in vitro assays for activation of the contact phasecomponent, for example, by measuring release of kinin or by looking atthe effect on coagulation times for example in a aPPT test, for example,as described below. Candidate substances may also be tested in vivo, forexample, as described below.

Assays

The assays in accordance with the invention may be in vitro assays or invivo assays, for example, using an animal model. In accordance with theinvention, the bacterial strain to be used in the assay may be anybacteria which is shown to interact with the contact phase system. Inparticular, strains of Streptococci, preferably S.pyogenes,curli-expressing bacteria such as E.coli and Salmonella and otherbacteria shown to bind any of the contact phase system components suchas Staphylococcus aureus are suitable candidates for use in the assaysof the invention.

The invention provides an assay having the following steps:

a) including a plasma sample with a bacterial strain,

b) adding the compound to be assayed

c) conducting an aPTT test on the sample; and

d) determining whether there is a decrease in the clotting time.

The invention has been described specifically with reference to an aPTTtest Kits for such tests are commercially available. Other tests whichwould also demonstrate a decrease in the clotting time may be used inaccordance with the invention. In particular, any test looking atcoagulation in which the presence of bacteria are shown to effect thecoagulation times would be a suitable test for use in accordance withthe present invention.

Plasma may be obtained from a mammalian source such as human or mouse.The bacterial strain can be incubated with a sample of plasma whichwould contain all of the contact phase components. The assay may also beconducted in the absence of one or more of the usual contact phasecomponents which would be present in plasma. Alternatively, the assaycould be carried out in the presence of those plasma components whichinteract with the bacteria and are required for normal clotting timesand any other components of the plasma alone which are necessary toconduct an aPTT test. Thus the assembly may be conducted by incubatingbacteria solely in the presence of the plasma components required forclotting. For example, the components of the intrinsic pathway ofcoagulation may be incubated with the bacteria.

Assays according to the invention may be carried out in a number ofdifferent ways. In particular, the assay could be carried out both inthe presence of a compound to be assayed and in the absence of thecompound to be assayed. A comparison between the two assays could thenbe made to determine the effect of the compound to be assayed todetermine whether that compound would be useful in the treatment ofbacteria disorders. Alternatively, as a control, the assay could becarried out in the absence of any bacteria. Thus, in accordance with oneaspect of the invention, the invention provides the steps of

incubating a plasma sample with a bacterial strain

adding a compound to be assayed to the sample

conducting an aPTT test on the sample

incubating a plasma sample with a bacterial strain and conducting anaPTT test on that sample

determining whether there is a difference in the clotting time in thetwo samples.

In general, it would be expected that the bacterial strain would havethe effect of increasing the clotting time, in particular the clottingtime in an aPTT test. A compound which may be useful in the treatment ofa bacterial induced coagulation disorder will have the effect ofdecreasing the clotting time when compared to the clotting time in theabsence of the compound. The compound itself may additionally have aneffect on the clotting time in the absence of bacteria and would stillbe useful in the treatment of bacterial induced coagulation disorders.

In an alternative aspect, the bacteria may be removed from the sampleprior to carrying out the aPTT test. The bacteria may be removed fromthe sample prior to addition of the compound to be assayed. In part,this is due to the ability of bacteria to deplete the contact phasecomponents from a sample. A compound which may be useful in thetreatment of a bacterial induced coagulation disorder may have theeffect of improving clotting times after such depletion of the contactphase components. Thus, in accordance with this aspect of the invention,the assay comprises the steps of incubating a plasma sample with abacterial strain, removing bacteria from the sample, adding the compoundto be assayed and conducting an aPTT test on the sample. These steps mayalso be carried out without including the step of adding the compound tobe assayed. The clotting times of the two tests may be compared toassess the effect of the compound under assay.

The compound may be added to the sample before, during or after thebacterial, since the compound may at in any way to alter the clottingtime. The assay can also be carried out as a challenge in an animalmodel with bacteria. Bacteria could be introduced into the animal toestablish survival of the animals following bacterial infection.Candidate substance can be co-administered with the bacteria orsubsequent to administration and prior to administration of bacteria toestablish whether the candidate substance can improve survival. It isparticularly preferred that animals will be challenged with bacteria.Blood samples can then be removed from the animal for use in the assaysin accordance with the invention. Thus, in this embodiment, an animal isfirst challenged with a bacterial strain,

a blood sample is taken from the animal,

a compound to be assayed is added to the blood sample

an aPTT test is carried out on the sample.

the clotting time is determined.

This assay could also be carried out without adding the compound underassay so that a comparison could be made to determine the effect of thecompound on the clotting time. It is also possible that the compoundcould be administered together with the bacteria so that a sample ofblood is then taken from the animal and the aPTT test carried out on theblood sample. In an alternative assay method, binding of the contactphase proteins to the surface of bacteria can be determined. Anyinhibitory effect by the candidate substance can then be evaluated. Onetype of in vitro assay for identifying substances which interfere withactivation of the contact phase proteins on the bacterial surfaceinvolves incubating bacteria in the presence of one or more contactphase components in the absence of a contact substance; incubatingbacteria with at least one contact phase component in the presence of acandidate substance; and determining if the candidate substance disruptsthe interaction between bacteria and the contact phase component.

Alternatively, bacteria can be incubated with at least two or all of thecontact phase components. Activation of the contact phase systemfollowing binding of the bacteria can be determined, for example, bymeasuring release of kinin from bound H-kininogen. As a control, theassay can be run in the absence of any bacteria. Similarly, anyinhibitory effect by the candidate substance can be evaluated.

In this assay, the candidate substance can be incubated together withthe bacteria and contact phase system. Alternatively, the bacteria mayfirst be incubated together with the elements of the contact phasesystem. Subsequently, the candidate substance can be introduced.

When assaying for binding of contact phase components, labels may beused such as a radioactive label, an epitope tag or an enzyme antibodyconjugated to the contact phase protein to determine the quantity ofbound protein to the bacteria surface. The effect of a candidatesubstance on an interaction between bacteria and the contact phasecomponents can be determined by comparing the amount of label bound inthe presence of contact candidate substance with the amount of labelbound in the absence of candidate substance. A lower amount of labelbound in the presence of the candidate substance indicates that thecandidate substance is an inhibitor of binding between the contact phaseproteins and bacteria.

Therapeutic uses

A key part of the bacterial infection process which in particular maylead to toxic shock like syndrome and coagulation disorders involvesactivation of the contact phase system on the surface of bacteria. Thepresent invention provides a substance capable of interfering withactivation of the contact phase system or substances which inhibit theeffects of activation of the contact phase system and provides the useof such substances in methods of treating bacterial infection.

The formulation of the substance according to the invention will dependupon the nature of the substance identified but typically a substancemay be formulated for clinical use with a pharmaceutically acceptablecarrier or diluent. For example, it may be formulated for topical,parenteral, intravenous, intramuscular, subcutaneous, intraocular ortransdermal administration. A physician will be able to determine therequired route of administration for any particular patient andcondition.

Preferably, the substance is used in an injectable form. It maytherefore be mixed with any vehicle which is pharmaceutically acceptablefor an injectable formulation, preferably from a direct injection at thesite to the treated. The pharmaceutically acceptable carrier or diluentmay be, for example, sterile or isotonic solution.

The dose of substance used may be adjusted according to variousparameters, especially according to the substance used, the age, weightand condition of the patient to be treated, to mode of administrationused and the required clinical regimen. A physician will be able todetermine the required route of administration and dosage for anyparticular patient and condition.

The invention will be described with reference to the following exampleswhich are intended to be illustrative only and not limiting. It has beenshown that kininogens bind to the surface of infectious bacteria such asStreptococcus pyogenes, Escherichia coli and Salmonella, and theseresults are presented in the appended examples 1 and 2. This bindingcauses activation of the so called contact phase system in which alsoprekallikrein, factor XI and factor XII participate. As a resultproteolytic enzymes called killikreins are activated, and these enzymesare able to release kinins from kininogens. Kinins are short peptides ofwhich bradykinin is the most important. These reactions areschematically outlined in FIG. 1.

Another microbial way of releasing kinins has also been found, and it isdescribed in detail in example 3. Streptococcus pyogenes produces acystein proteinase (se e.g. WO 96/08569) which directly can degradeH-kininogen thereby releasing physiologically active kinins. ThusStreptococcus pyogenes can induce release of kinis not only on thebacterial surface by activating the contact phase system, but also byproducing SCP which is able to directly degrade H-kininogen in the bloodstream.

As already mentioned bradykinin and other kinins are potential peptidehormonescausing fever, hypotension, increased vascular permeability,contraction of smooth muscles and pain. A massive release of kinis maytherefore explain many of the symptoms observed during severe infectiondiseases.

By blocking the kinin effect with kinin antagonists these diseasesymptoms can be cured as is shown in tests on mice which are presentedin example 4. These tests show that mice infected with lethal doses ofS. pyogenes or of SCP live longer and look healthy and unaffected ifthey are treated with the bradykinin antagonist HOE 140 (Boa et al.(1991), Eur. J. Pharmacol., vol. 200, pp. 179-182).

The invention accordingly relates to use of kinin antagonists forpreparing a pharmaceutical composition for treating and preventingbacterial infections. All pharmaceutically acceptable substances capableof preventing the physiological actions of kinins can be used accordingto the present invention. The kinin antagonists HOE 140 (Bao et al.,supra), NPC 17751 (Mak et al. (1991), Eur. J. Pharmacol., vol. 194, pp.37-43), NPC 349 (Wirth et al. (1995), Can. J. Pharmacol., vol. 73, pp.797-804), CP0127 (Whalley et al. (1992), Agents, Actions Suppl., vol.38(Pt3):pp. 413-20), NPC-1776 (Cheronis et al., eds.: Proteases,Protease Inhibitors and Protease-Derived Peptides (1993, BirkhauserVerlag, Basel, C11), pp. 167-176), WIN 64338 Sawutz et al (1995), Can.J. Physiol. Pharmacol., vol. 73, pp. 805-811), des-Arg9-[Leu81bradykinin (Pruneau et al. (1996), Eur. J. Pharmacol, vol. 297 pp.53-60), DesArg9-D Arg[Hyp3,Thi5,D-Tic7,Oic8]-bradykinin(desArg10-[HOE140]) (Wirth et al. (1991), Eur. J. Pharmacol., vol. 205,pp. 217-218) and Sar4-[D-Phe8]-des-Arg9-bradykinin (Gouin et al. (1996),vol. 28, pp. 337-43) and other suitable kinin antagonists disclosed inthese articles are especially preferred. The antagonists can be usedagainst infections caused by Gram positive and/or Gram negativebacteria. Ft is preferred to use them against infections caused bybacteria belonging to the genera Streptococcus., scherichia, Salmonella,Staphylococcus, Klebsiella, Moracella, Haemophilus and Yersinia.

Suitable pharmaceutical compositions comprises an effective amount ofthe kinin antagonist or a pharmaceutically acceptable derivative or saltthereof and one or more pharmaceutically acceptable inert carriers orexcipients.

The compositions can be used for treating any warm blooded animalincluding humans.

The amount of the active compound may vary from 0.001% to about 75% byweight of the composition. Any pharmaceutically acceptable inertmaterial which does not degrade or otherwise react with the antagonistcan be used as a carrier.

The pharmaceutical compositions are prepared in a manner well-known tothe skilled person. The carrier or excipient may be a solid, semisolidor liquid material which can serve as a vehicle or medium for the activeingredient.

The composition may be administered in any form or mode which makes thecompound bioavailable in effective amounts, including oral andparenteral routes. It can, for example, be administered orally,subcutaneously, intramuscularly, intravenously, transdermally,intranasally, and rectally. Oral administration is preferred.

The active compound can be administered in the form of tablets,capsules, suppositories, solutions, suspensions etc.

The carrier may be an inert diluent or an edible carrier. The adjuvantsmay be binders such as microcrystalline cellulose, gum traganth,gelatine, starch or lactose; disintegrating agents such as alginic acid,primogel, corn starch; luricants such as magnesium stearate; glidantssuch as colloidal silicon dioxide, sweetening agents such as sucrose orsaccharin or a flaving agent; liquid carriers such as polyethyleneglycol or a fatty oil.

Tablets or pills may be coated with sugar, shellac or other entericcoatings.

Solutions or suspensions may also include sterile diluents such as waterfor injection, saline solution, fixed oils, polyethylene glycols,glycerine, propylene glycol or other synthetic solvents; chelatingagents; buffers and agents for adjusting tonicity, such as sodiumchloride or dextrose.

The invention will now be more closely described in the followingexamples.

EXAMPLE 1

M proteins are fibrous, hair-like structures expressed at the surface ofStreptococcus pyogenes (Fischetti (1989), Clin. Microbiol. Rev, Vol 2,pp 285-314; Kehoe (1994), New Compr. Biochem., vol. 27, pp. 217-261).They are regarded as major virulence determinants due to theirantiphagocytic property, and exist in more than 80 different serotypes.A role for M protein in fibrinolysis was suggested by the observationthat some M proteins bind plasminogen (Berge et al. (1993), J. Biol.Chem. vol. 268, pp. 25417-25424).

S pyogenes is an important human pathogen causing common suppurativeinfections such as pharyngitis and skin infections, but also hyperacuteand life-threatening toxic conditions as weft as serious post-infectiousconditions (rheumatic fever and glomerulonephritis).

Bacterial strains and culture conditions.

S. pyogenes strains AP 1 (40/5 8) and AP46 (1/59), expressing the M 1and M4 proteins, respectively, and the M negative strain AP74 (30/50)were from the Institute of Hygiene and Epidemiology, Prague, CzechRepublic. Bacteria were grown in Todd Hewitt, broth (Difco) at 37° C.for 16 hours, washed twice in PB S (0. 15 M NaCl, 0. 02 M phosphate, pH7.4) containing 0.02% NaN₃ (w/v) (PBSA), and resuspended in PBSA to2×10¹⁰ cells/ml. Cells from this stock suspension were used in plasmaabsorption experiments and kallikrein digestions assays.

Proteins and labeling of proteins

Kininogens were purified from human plasma as previously described(Muller Esterl et al. (1988), Methods Enzymol., vol. 163, pp. 240-256).The monoclonal antibody HKL 16 directed to an epitope in domain 6(domain D6H) of the light chain of HK was raised in mouse and affinitypurified on protein A-Sepharose (Kaufmann et al.(1993), J. Biol. Chem.vol. 268, pp. 9079-9091). The monoclonal antibody HKL 19, also directedagainst the D6H domain of HK, has been described (Kaufmann et al.(1993),J. Biol. Chem., vol. 268, pp. 9079 9091). Prekallikrein was isolatedfrom human plasma (Hock et al. (1990), J. Biol. Chem. vol.265, pp.12005-12011). Human factor XI was a kind gift of Dr. J. Meijers,Department of Hermatology, University of Utrecht, The Netherlands. Humanfactor XII was from Enzyme Research Laboratories, South Bend, Ind., USARecombinant M1 and M46 proteins were produced as described (Berge etal., supra; Akesson et al. (1994), Biochem. J. vol. 877-886).

Plasma absorption Experiments

The plasma absorption experiments were performed as described (Sjobringet al.(1994), Mol. microbiol. vol. 14,pp. 443-452). Fresh human plasmawas mixed with proteinase inhibitors (Sigma Chemical) to a finalconcentration of 2 mM aprotinin, 0.1 mM diisopropylfluorophosphate(DFP), 1 mM soy bean trypsin inhibitors (SBTI), 0.1 mMphenylmethylsulfonylfluoride (PMSF), and 5 mM benzamidine chloride.Bacteria (2×10¹⁰ cells/ml) suspended in 1 ml PBST (PBS containing 0.05%Tween 20) were incubated with 1 ml human plasma for 60 min at 37° C. Thecells were pelleted and washed three times with 10 ml of PBST. To elutethe absorbed proteins, the bacteria were incubated for 5 min with 0.5 mlof 30 mM HCl, pH 2.0. The bacteria were pelleted and the supernatant wasimmediately buffered with 50 μl of 1M Tris, concentrated to 250 μl (witha buffer change to PBS) by ultrafiltration on an Amicon Centricon-30(Amicon Inc. Beverly, Mass. U.S.A.). Twenty microliters of the proteinsolution was analysed by SDS-PAGE and immunoprint experiments using themonoclonal antibody HKL16.

ELISA

The Sandwich ELISA technique was performed as detailed (Muller-Esterl etal. (1988), supra; Kaufmann et al. (1993), supra; Kaufmann (1993), Annu.Rev. Immunol. vol. 11, pp. 129-163) with modifications as follows;microtiter plates (Immunolon, Dynatech) were coated with 1 μg/ml of themonoclonal antibody HKL19 in 15 mM NaHCO₃/Na₂CO₃ buffer, pH 9.6,containing 1.5 mM NaN₃ by overnight incubation at 4° C. The plates werewashed five times with 20 mM phosphate buffer, pH 7.4, containing 150 mMNaCl and 0.05% (w/v) Tween 20 (PBST). Serial doubling dilutions of HKstandards (starting at a concentration of 2 μg/ml) or samples wereprepared in PBST containing 2% BSA and 200 μl were added to the coatedwells in duplicate. After incubation for 1 hour at 37° C. and washing asabove, 200 μl of a polyclonal anti-HK sheep IgG diluted 1:1000 in PB STcontaining 2% BSA were applied per well followed by incubation for 2hours at 37° C. The plates were extensively washed (see above) and 200μl of a POD-conjugated antisheep secondary antibody was added andincubated for 2 hours at 37° C. Finally, after washing as above, 200 μlof 0.1% (w/v) ABTS(2,2′azino-bis(3-ethyl2,3-dihydrobenzthiazoline)-6-sulfonate), 0.05%(v/v) H₂O₂ in 0.1 citric acid, 0.1 M NaHPO, were added to each well andthe plates were incubated for 30 min at 37° C. in the dark. The changein absorbance at 405 nm was measured, and the amounts of HK in thesamples determined using the HK standard curve.

Electrophoresis and electroblotting

SDS-PAGE (polyacrylamide gel electrophoresis) was performed as described(Neville (1971), J. Biol. Chem., vol. 246, pp. 6328-6334). Proteins ineluates from absorption experiments with bacteria were separated on gelsof 10% total acrylamide with 3% Bisacrylamide. Before loading, sampleswere boiled for 3 min in sample buffer containing 2% SDS and 5%β-mercaptoethanol. For Western blotting analysis, proteins weretransferred to polyvinylidenedifluoride (PVDF) membranes (Immobilon,Millipore) by electroblotting from gels as described (Towbin et al.(1979), PNAS, vol 76, pp. 4350-4354) using a Trans Blot semi-drytransfer cell (Bio-Rad, Irvine, Calif., U.S.A.).

Determination of affinity constants

Binding kinetics were determined by surface plasmon resonancespectroscopy using a BIAlite biosensor system (Pharmacia Biosensor AB,Freiburg, Germany). HK was immobilised on research grade CM5 sensorchips in 10 mM sodium acetate, pH 4.5, using the amine coupling kitsupplied by the manufacturer. All measurements were carried out inHEPES-buffered saline that contained 10 mM HEPES, pH 7.4, 150 mM NaCl,3.3 mMEDTA, and 0.005% Surfactant P.20 (Pharmacia Biosensor AB).Analyses were performed at 25° C. and at a flow rate of 10 μl/min. tocalculate affinity constants, 30 μl of streptococcal M1 protein and M46protein were applied in a serial dilution (starting concentration 100μg/ml) followed by injection of buffer alone (30 μl). In addition, 30 μlof plasmakallikrein samples (doubling dilution; starting concentrationof 25 μg/ml) were assayed for comparison. Surfaces were regenerated with30 μl of 10 mM HCl at a flow rate of 10 μl/min. The kinetic data wereanalysed by the BIA evaluation 2.0 program (Pharmacia Biosensor AB).Alternatively, 30 μl of M1 protein (50 μg/ml) and plasmakallikrein (10μg/ml) alone or in combination were added and the amount of boundprotein was determined, expressed in resonance units (RU) according tothe manufacturer's descriptions.

Immunoprint analysis of HK and its fragments

Immunoprint analysis was performed as follows. Briefly, the PVDFmembranes were blocked in PBST containing 5% (w/v) nonfat dry milk for20 min at 37° C. (Timmons et al. (1990), Methods Enzymol. vol. 182, pp.679-688), washed three times with PBST for 5 min and incubated withantibodies against HK (in the blocking buffer) for 30 min at 37° C.After washing the sheets were incubated with peroxidase-conjugatedsecondary antibodies for 30 min at 37° C. Secondary antibodies weredetected by the chemiluminescence method (Timmons et al. (1990), supra;Nesbitt et al. (1992), Anal. Biochem., vol. 206, pp. 267-272).Autoradiography was done at room temperature for 1-2 min using KodakX-Omat S films and Cronex Extra Plus intensifying screens.

Kallikrein digestion assay and quantification of released bradykinin

Plasma prekallikrein was activated to plasma kallikrein by cleavage withactivated factor XIIa as described (Berger et al. (1986), J. Biol.Chem., vol. 261, pp. 324-327). Briefly, plasma prekallikrein was mixedwith factor XIIa in a molar ratio of 10:1 in PBS, pH 7.4, and incubatedfor 2 hours at 37° C. and immediately used for proteolysis experiments(see below). Bacteria (4×10¹⁰ cells) in 0.5 ml PBST were incubated with1.5 ml human plasma diluted 2:1 in PBST containing protease inhibitors(see Absorption experiments). After incubation for 1 hour at 37° C. thecells were washed three times in 10 ml PBST and resuspended in 0.4 mlkallikrein assay buffer, 0.15 m Tris-HCl, pH 8.3 (Hock et al. (1990),supra). Bacteria were mixed with 100 μl of plasma kallikrein (giving afinal enzyme concentration of 0.02 μg/ml) in an Eppendorf tube andincubated for 10 min at 37° C. An equal amount of bacteria suspensionwas incubated, as a control, with 10 μg kallikrein assay buffer withoutkallikrein. The incubation was ended by adding 0.4 ml of 60 mM HCl.Cells were pelleted and the supernatants were immediately buffered byadding 100 μl of 1 M Tris and ultrafiltrated through an AmiconCentricon-10 filter (Amicon Inc.) to separate free BK from kininogenfragments. The resulting filtrates were analysed for their BK content bysolid phase radioimmunoassay using the MARKIT-M bradykinin TM kit(Dainippon Pharmaceutical Co., Osaka, Japan).

Results

HK is absorbed from human plasma by M protein-expressing S. pyogenesbacteria

M proteins are known to interact with a number of different plasmaproteins including fibrinogen, plasminogen, immunoglobulins, albumin,and factor H and C4 binding protein of the complement system (Kehoe(1994), supra). This raises the question whether the interaction betweenHK and M protein can take place in the presence of these other M proteinbinding plasma proteins, especially as proteins like albumin, IgG, andfibrinogen occur at much higher concentrations than HK (HK representsapproximately 0.15% of the total protein content in plasma). To addressthis question two strains of S. pyogenes expressing M1 and M46 protein,respectively, were incubated with fresh human plasma. Followingextensive washing, proteins bound to the bacterial surface were elutedand the amount of HK was determined by ELISA. In a representativeexperiment the amounts of HK eluted from M1 and M46 bacteria were 63.6and 123.6 pmol/10 ¹³ bacteria, respectively, whereas an M proteinnegative mutant strain (AP74) absorbed HK at background level (below 10pmol/10 ¹² bacteria). The results demonstrate that HK also in a plasmaenvironment can interact with M protein-expressing S. pyogenes bacteria.

Analysis of the binding of M proteins to HK suggests that HK isaccumulated at the streptococcal surface

Using surface plasmon resonance spectroscopy, the binding kineticsbetween HK and the M1 and M46 proteins were determined and compared tothe interaction between HK and plasma prekallikrein. In theseexperiments different amounts of purified M proteins or plasmaprekallilrein were applied and left to interact with immobilised HK tothe level of saturation. FIG. 4 shows typical sensorgrams obtainedbetween HK and M1 protein (4A) and plasma prekallikrein (4B). Theresults obtained for HK-M46 (not shown) were similar to those for HK-M1shown in FIG. 4A. On the basis of these experiments, dissociation andassociation rates were calculated, which when divided give thedissociation constants (Table 1). The figures demonstrate that thedissociation of M1 and M46 proteins are slower than their association,permitting an accumulation of HK at the bacterial surface. It can alsobe noted that the HK affinity is higher for M46 than for M1 protein. Inplasma, prekallikrein circulates in complex with HK. As shown in FIG. 4Band Table 1, both the association and dissociation rates are high forthe interaction between these proteins. However, different data suggestthat the binding of M proteins to HK is not disturbed by the interactionbetween plasma prekallikrein and HK. Thus, prekallikrein interacts withHK through the D6H domain, residues 569-595 (Berger et al. (1986),supra), whereas the binding site for M1 protein in HK is in domain D5H,residues 479-496 (Ben Nasr et al. (1996), Mol. Microbiol. vol. 14, pp.927-935). Moreover, when a sample containing both M1 protein andprekallikrein was applied to an HK-coupled sensor chip, a binding of 909RU was recorded (FIG. 4A) which represents almost 100% of the calculatedmaximum binding. In other experiments the binding of plasmaprekallikrein to preformed complexes between HK and M1 protein wasstudied (FIG. 4B). The results of these experiments also suggest that M1protein and plasma prekallikrein can bind simultaneously to HK. Finally,this is also supported by the fact that plasma prekallikrein in anindirect ELISA (Berger et al. (1986), supra) was absorbed by HK bound toimmobilised M1 protein (data not shown).

HK is cleaved at the surface of S. pyogenes

Using antibodies against the light chain of HK in Western blotexperiments, we analyzed the HK bound to and eluted from Mprotein-expressing bacteria. Intact HK has a molecular mass of 121 kDaand the release of the BK nonapeptide from HK results in the formation atwo-chain molecule with a heavy and a light chain (63 and 58 kDa,respectively) connected by a disulfide bond. The light chain is in theCOOH-terminal part of HK and as shown in FIG. 5 the antibodies againstthis chain bind to two bands corresponding to the light chain of 58 kDa,and a 45 kDa fragment of the light chain. No band is seen at the placefor HK (121 kDa), demonstrating an efficient cleavage of HK at thebacterial surface. These results 18 suggest that HK is released as aconsequence of the binding of HK to the streptococci, as HK in plasmaoccurs only in its intact form (Silverberg et al. (1988), MethodsEnzymol., vol. 163, pp. 85-95). Moreover, HK in plasma incubated withthe M-protein-negative S. pyogenes strain AP74 (Ben Nasr et al. (1995),Biochem. J, vol. 305, pp. 173-180) showed no degradation, a result whichwas not affected by the presence or absence of proteinase inhibitors inthe plasma sample. The step-wise proteolysis of HK leading to BK releasestarts with the generation of a heavy chain in which the bradykininpeptide is still attached at the COOH-terminal end. Secondary cleavageby activated plasma prekallikrein then releases the BK peptide. Whenactivated plasma prekallikrein was added in excess to the plasmaproteins bound to the streptococci, BK was released and quantified. Thelevel of BK release from strains AP1 and AP46 were 10.7±2.7 and 19.8±7.9pmol/10¹² bacteria, respectively (values are the means of threeexperiments=1 standard deviation). However, in parallel experimentswhere activated prekallikrein was added to AP74 bacteria preincubatedwith human plasma, no release of BK was detected.

TABLE 1 Affinity rates and dissociation constants for the interactionsbetween immobilised HK and M1 protein, M46 protein, or plasmaprekallikrein* dissociation association rate dissociation rate constantligand (10³ × s⁻¹ M⁻¹⁾ (10⁻³ × s⁻¹⁾ (10⁻⁴ × M) MI protein 5.5 ± 0.8 3.7± 0.7 68.3 ± 15.7 M46 protein 9.5 ± 2.1 2.3 ± 0.3 24.8 ± 5.9 Prekallikrein 496 ± 128 18.3 ± 5.4  3.8 ± 1.3 *Values are mean ± SEMfrom at least three different experiments.

Example 2

Assembly of human contact phase proteins and release of bradykinin atthe surface of curli-expressing Escherichia coli

In the present invention, a number of bacterial strains, belonging toseveral species and isolated from patients with sepsis, are tested asconcern their different abilities to bind HK. In addition, theinteraction between factors of the contact phase system and E. coli isanalysed.

Bacterial strains and culture conditions

108 bacterial strains of human sepsis origin were isolated at theDepartment of medical Microbiology, Lund University, Lund, Sweden. SixStreptococcus pyogenes strains from patients with sepsis, isolated inSweden between 1980-89, were kindly provided by Dr. Stig Holm UmeaUniversity, Umea, Sweden. Seventeen E. coli strains causing differentgastrointestinal infections were kindly provided by Dr. James P. Nataro,from the Center for Vaccine Development University of Maryland School ofMedicine, Baltimore, Md. The strains are designated EHEC 1-4 (84-7025;83-8006; 78-534; 84-7453); EIEC 1-4 (IC651-1; 1C711-1; EI705-1;EI439-1). EPEC 1-4 (147-150983; 2348/69; 2087-77; 2395-80), ETEC 1-5(EI142-4; E17-10; EI123-3; EI166-9; EI150-9) YMel is a curli-expressingE. coli K12 strain (Richenberg, H. V., and Lester, G. (1955) Thepreferential synthesis of beta-galactosidase in Escherichia coli. J GenMicrobiol 13: 279-284,1955). YMel-1 is an isogenic curli-deficientmutant strain generated by insertional mutagenesis of the csgA gene inYMel (Olsen, A, Arnqvist, A., Hammar, M., Sukupolvi S., and Normark, S.(1993) The RpoS sigma factor relieves H-NS-mediated transcriptionalrepression of csgA, the subunit gene of fibronectin binding curli inEscherichia coli, Mol Microbiol 7: 523-536). All Gram-positive speciesand the Moraxella catarrhalis strains were grown in Todd-Hewitt broth(Difco) at 37° C. for 16 hours. Haemophilius influenzae, Neisseriameningitidis were grown on brain-heart infusion supplemented with Haeminand nicotinamid. All other Gram-negative species were grown onLuria-Bertani (LB) broth for 16 h at 37° C. In some experiments,Salmonella enteritidis and E. coli species, designated ES 1-7 (for E.coil sepsis isolate number 1-7), were grown on LB- or colonisationfactor agent (CFA)-agar (Evans, D. G., Evans, J. D. J., and Tjoa, W.(1977) Hemagglutination of human group A erythrocytes by enterotoxigenicEscherichia coli isolated from adults with diarrhoea: correlation withcolonization factor, Infect Immun 18: 330-337) at 26° C. forapproximately 40 h at 26 or 37° C. Bacteria were washed twice in PBS(0.15 M NaCl 0.06 M phosphate, pH 7.2) containing 0.02% NaN₃ (w/v)(PBSA), and resuspended in PBSA to 2×10¹⁰ cells/ml Cells from this stocksuspension were used in protein binding assays or in plasma absorptionexperiments.

Proteins and labeling of proteins

Kininogens were purified from human plasma as previously described(Muller-Esterl W., Johnson, D A, Salvesen, G., and Barrett, A. J. (1988)Human kininogens. Methods Enzymol 163: 240-256). The monoclonal antibodyHKL16 directed to an epitope in domain 6 (domain D6H) of the light chainof HK was raised in mouse and affinity purified on protein A-Sepharose(Kaufmann, L, Haasemann, M., Modrow, S., and Muller-Esterl, W. (1993)Structural dissection of the multidomain kininogens. Fine mapping of thetarget epitopes of antibodies interfering with their functionalproperties. J Biol Chem 268: 9079-9091). The polyclonal anti-bradykininantibodies, α-BK (AS348) were raised in rabbit using a conjugate ofbradykinin and keyhole limpet hemocyanin covalently coupled by thecarbodiimide method. Prekallikrein was isolated from human plasma (Hock,L, Vogel, R., Linke, R-P., and Muller Esterl, W. (1990) High molecularweight kininogen-binding site of prekallikrein probed by monoclonalantibodies. J Biol Chem 265: 12005-12011). Human factor XI was a kind ofDr. J. Meijers, Department of Hematology, University of Utrecht TheNetherlands. Human factor XII was from Enzyme Research Laboratories,South Bend, Ind., U.S.A. Bovine serum albumin (BSA), human serum albumin(HSA), fibrinogen, fibronectin, and plasminogen were from Sigma Chemical(St. Louis, Mo.). Purification of curli from the E coli strain YMel wasperformed as described (Collinson, S. K., Emody, L., Muller, K.-H.,Trust, T. L, and Kay, W. W (1991) Purification and characterization ofthin, aggregative fimbriae from Salmonella enteritidis. JBacteriol 173:4773-4781; Olsen, A. X., Hanski E., Normark, S., and Caparon, M. G.(1993) Molecular characterization of fibronectin binding proteins inbacteria. In J Microbiol. Methods. Boyle, M. D. P. (ed). London:Elsevier Science Publisher, pp. 213-226). The M1 protein and thePCR-generated M1 protein fragment (S-C3) were produced as described(Akesson, P, Schmidt, K.-H., Cooney, L, and Bjorch L. (1994) M1 proteinand protein H: IgGFc- and albumin-binding streptococcal surface proteinsencoded by adjacent genes. Biochem J300: 877-886). Proteins were labeledwith I¹²⁵ using the Bolton and Hunter reagent (Amersham Corp., U.K.).

Bacterial binding assay

Bacteria were resuspended in PBSA containing 0.05% (w/v) of Tween-20(PBSAT) and the cell concentration was adjusted to 2×10¹⁰ cells per ml.Bacteria were diluted as indicated, and 200 μl of the resultingsuspensions was incubated with radiolabeled protein in 25 μl PBSAT (10⁴cpm) at 37° C. for 60 min. For screening of the different bacterialisolates, dilutions of 2×10⁹ cells/ml were used. After incubation two mlof PBSAT was added, the cell suspension was centrifuged, and theradioactivity of the pellet was counted. The binding activity wasexpressed as the percentage of the total radioactivity added per tube.

Absorption experiments

The plasma absorption experiments were performed as described (Sjobring,U., Pohl, G., and Olsen, A. (1994) Plasminogen, absorbed by Escherichiacoli expressing curli or by Salmonella enteritidis expressing thinaggregative fimbriae, can be activated by simultaneously capturedtissue-type plasminogen activator (t-PA), Mol Microbiol 14: 443-452)with some modifications. Fresh human plasma was mixed with proteaseinhibitors (Sigma Chemical) to a final concentration of 2 mM aprotinin,0.1 mM diisopropylfluorophosphate (DFP), 1 mM soy beam trypsininhibitors (SBTI), 0.1 mM phenylmethylsulfonylfluoride (PMSF) and 5 mMbenzamidine chloride. Bacteria (2×10¹⁰ cells/ml) suspended in 1 ml PBST(PBS containing 0.05% Tween 20) were incubated with 1 ml human plasmafor 60 min at 37° C. The cells were pelleted and washed three times with10 ml of PBST. To elute the absorbed proteins, the bacteria wereincubated for 5 min with 0.5 ml of 30 mM HCl, pH 2.0. The bacteria werepelleted and the supernatants was immediately buffered with 50 μl of 1MTris, concentrated to 250 μl (with a buffer change to PBS) byultrafiltration on an Amicon Centricon-30 (Amicon Inc. Beverly, Mass.,U.S.A.). Ten microliters of the protein solution was analysed bySDS-PAGE and immunoprint experiments using antibodies to HK orbradykinin.

Electrophoresis and electroblotting

SDS-PAGE was performed as described (Neville, D. M. J. (1971) Molecularweight determination of protein-dodecyl sulphate complexes by gelelectrophoreses in a discontinuous buffer system. J Biol Chem 246:6328-6334). Plasma proteins and the proteins in eluates from absorptionexperiments with bacteria were separated on gels of 10% total acrylamidewith 3% Bis-acrylamide. Before loading, samples were boiled for 3 min insample buffer containing 2% SDS and 5% mercaptoethanol. Curli-subunitswere separated on gels of 13.6% total acrylamide. Curli-containingpreparations were not boiled prior to electrophoresis. For Westernblotting analysis, proteins were transferred to polyvinylidenedifluoride(PVDF) membranes (Immobilon, Millipore) by electroblotting from gels asdescribed (Towbin, R., Staehelin, T., and Gordon, J. (1979)Electrophoretic transfer of proteins from polyacrylamide gels tonitrocellulose sheets: Procedure and some applications. Proc Natl AcadSci USA 76: 4350-4354) using a Trans Blot semi-dry transfer cell(BioRad, Irvine, Calif., U.S.A.). The membranes were blocked and washedat 37° C., and incubated with [¹²⁵I]-labeled proteins as described(Sjobring et al., 1994, op. cit). Autoradiography was done at −70° C.using Kodak X-Omat S films and Cronex Xtra Plus intensifying screens.

Immunoprint analysis of HK and its fragments

For immunoprint analysis of the electrotransferred proteins followingplasma absorption experiments, the PVDF membranes were blocked in PBSTcontaining 5% (w/v) nonfat dry milk for 20 min at 37° C. (Timmons, T.M., and Dunbar, S. D. (1990) Protein blotting and immunodetection.Methods Enzymol 182: 679-688), washed three times with PBST for 5 minand incubated with antibodies against HK (from mouse or rabbit, 2 μg/mlin the blocking buffer) for 30 min at 37° C. After washing the sheetswere incubated with peroxidase-conjugated secondary antibodies againstmouse or rabbit IgG (rabbit anti-mouse and sheep anti-rabbit,respectively) for 30 min at 37° C. Secondary antibodies were detected bythe chemiluminescence method (Nesbitt, S. A., and Horton, M. A. (1992) Anon-radioactive biochemical characterization of membrane proteins usingenhanced chemiluminescence. Anal Biochem 206: 267-272). Autoradiographywas done at room temperature for 1-2 min using Kodak X-Omat S films andCronex Extra Plus intensifying screens.

Competitive binding, assay and affinity constant determination

Constant amounts of purified, polymeric curli (Olsen et al., 1993,op.cit) and [¹²⁵I-HK were incubated with varying amount of unlabeled HK.All reagents were diluted in incubation buffer (PBST containing 0.25%(w/v) BSA) to a total volume of 250 μl, incubated for 3 hours at 37° C.under gentle shaking, washed with incubation buller, and centrifuged for15 min at 3000 g. The radioactivity of the resulting pellet was measuredand the affinity constant was calculated as described (Akerstrom, B.,and Bjorck, L. (1989) Protein L: an immunoglobulin light chain-bindingbacterial protein. Characterization of binding and physicochemicalproperties. JBiol Chem 264: 19740-19746) using the formula of Scatchard(Scatchard, G. (1949) The attractions of proteins for small moleculesand ions, Ann N YAcad Sci 51: 660-672).

Kallikrein digestion assay

Plasma prekallikrein was activated to plasma kallikrein by cleavage withactivated factor XIIa as described (Berger, D., Schleuning, W. D., andSchapira, M. (1986) Human plasma kallikrein. Immunoaffinity purificationand activation to α- and β-kallikrein. JBiol Chem 261: 324-327).Briefly, plasma prekallikrein was mixed with factor XIIa in a molarratio of 10:1 in PBS, pH 7.4, and incubated for 2 hours at 37° C. andimmediately used for proteolysis experiments (see below). Bacteria(5×10¹⁰ in 0.5 ml PBST were incubated with 1.5 ml human plasmacontaining protease inhibitors (see “Absorption experiments”). Afterincubation for 1 hour at 37° the cells were washed three times in 10 mlPBST and resuspended in 0.5 ml kallikrein assay buffer, 0.15 m Tris-HCl,pH 8.3 (Hock et al, 1990, op. cit). Aliquots of 100 μl bacterialsuspension in Eppendorf tubes were incubated with plasma kallikrein for5, 10, 20 and 40 min at 37° C. One sample was incubated in the absenceof kallikrein for 40 min at 37° C. At indicated times, 0.5 ml of 100 mMHCl was added to the mixtures, and the samples were incubated for 5 minat room temperature to allow the absorbed proteins to dissociate. Thecells were pelleted and the supernatant was immediately buffered byadding 50 μl of 1 M Tris. The supernatants, were concentrated to 50 μl(with a buffer change to PBS) by ultra filtration on an AmiconCentricon-30 (Amicon Inc.). Twenty μl of the protein solution wasanalysed by SDS-PAGE followed by Western blotting and immunostainingusing the polyclonal anti-bradykinin antibodies (α-BK).

Results

Several pathogenic bacterial species bind kininogens

It has previously been demonstrated that most strains of the humanpathogen Streptococcus pyogenes bind kininogens through M proteins, afibrous surface protein and virulence determinant. According to thepresent invention, it has now been shown that strains of several otherpathogenic bacterial species, both Gram-positive and Gram-negative,isolated from patients with sepsis, also bind kininogens, especiallyH-kininogen (HK).

A total of 118 bacterial strains, all isolated from patients with sepsisand belonging to 18 different species, were tested for binding ofradiolabeled HK and LK at pH 7.2 in PBS. HK and LK share theirNH₂-terminal domains D1-D4, whereas they diller in their COOH-terminaldomains (see Miffier-Esterl et al., 1988). The majority of β-haemolyticstreptococcal strains and E. coli and Salmonella enteritidis strainsbound more than 25 percent of added M whereas many strains ofStaphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae,Moraxella catarrhalis, Haemophilus influenzae, and Yersiniaenterocolitica bound 10-25 percent. Strains of the remaining specieswere mostly negative (binding below 10 percent). All the strains werealso tested for binding of LK. Apart from some of the E. coli isolateswhich bound up to 40 percent the other strains showed low or backgroundinteraction. It was observed that culture conditions affected thebinding of HK to E. coli and S. enteritidis. Thus, binding activitieswere clearly higher when the strains were tested following growth at 26°C., as compared to bacteria grown at 37° C. Binding studies wereconducted both at 37° C. and 20° C., but in this case there was nosignificant difference. The high binding of HK to most E. coli strainsprompted us to test additional E. coli isolates from patients withsepsis and different gastrointestinal infections. High binding of HK wasfound among enterohaemorrhagic, enterotoxigenic and sepsis strains,whereas enteroinvasive and enteropathogenic strains were negative (FIG.8). Following growth at 37° C. binding was clearly lower with maximumbinding activities around 20 percent. However, the binding pattern wasthe same.

HK binding strains of E. coli absorb HK from human plasma

To investigate if E. coli bacterial which bind purified HK also take upkininogen from plasma, six strains previously analyzed for binding ofradiolabeled HK (strains EHEC 1, EHEC3, EIEC 1, EPEC 1, ETEC5, ES 1 ofFIG. 8) were incubated with human plasma for 60 min at 37° C. Thebacteria were washed and the plasma proteins bound to the cells weresubsequently eluted with pH 2.0. Following centrifugation proteinspresent in the supernatant were subjected to SDS-PAGE and Western blotanalysis (FIG. 8). The monoclonal antibodies used (HKL16) in theseexperiments detect an epitope in the light chain of HK (see FIG. 2), andas shown in the blot of FIG. 8, the strains that bind radiolabeled HK(EHEC3, ETEC5, ES 1) also absorb HK from human plasma, whereas thenon-binding strains (EHEC 1, EIEC 1, EPEC 1) fail to pick up HK fromplasma. Due to the qualitative nature of the immunoprint technique, weare unable to assess the fraction of plasma HK absorbed by the bacteria.The bands reacting with the antibodies correspond to single-chain HK(120 kDa), the intact light chain of HK (58 kDa), and a degraded form ofthe HK light chain (45 kDa). These results demonstrate that E. colistrains binding radiolabeled M also bind HK in a complex mixture ofproteins such as plasma (HK represents approximately 0.15% of the totalprotein content in plasma).

Curli mediate the binding of HK to E. coli

To test whether curli were involved in the binding of thecurli-expressing E. coli K12 strain YMel and an isogenic curli deficientmutant strain, YMel-1, were used in binding experiment with [¹²⁵I]-HK.As shown in FIG. 9 (left panel), only curliated Ymel bacteria showedaffinity for HK in a concentration-dependent manner.Transcomplementation of the curli-negative YMel-1 strain with a plasmidcontaining the structural gene for the curli subunit (csgA) restoredHK-binding. Plasma absorption experiments performed with differentconcentrations of YMel bacteria, demonstrated that the amount of HKabsorbed by the bacteria was dependent on the number of curliated cellsused (FIG. 9, right panel). In these experiments a constant volume ofplasma was used, and non-curliated YMel-1 cells were added to give thesame total number of bacteria for each absorption experiment. Binding ofHK to curli was also demonstrated with purified proteins. RadiolabeledHK was found to bind to monomeric curli subunits directly applied toPVDF membranes (not shown). Curb monomers separated by SDS-PAGE andblotted to PVDF membranes also reacted with [¹²⁵I]-HK (FIG. 10, lane A).In these experiments, intact HK-binding streptococcal M1 protein, and anon-binding COOH-terminal fragment (S-C3) of M1 (Ben Nasr, A., Herwald,H., Muller-Esterl, W., and Bjorck, L. (1995) Human kininogens interactwith M protein, a bacterial surface protein and virulence determinant.Biochem J 305: 173-180), were included as positive and negativecontrols, respectively (FIG. 12, lanes B and C). The immunoprintrevealed that M1 protein binds to HK as did curli monomers, but not theS-C3 fragment. Unlabeled HK completely blocked the binding of[¹²⁵I]-labeled HK to curliated E. coli as well as to isolated curli. InScatchard plots, the affinity constant for the interaction between HKand purified polymeric non-soluble curli was determined to be 9×10⁷ M⁻¹(FIG. 11). Fibronectin and plasminogen, other ligands for curli (Olsenet al., 1989; Sjobring et al., 1994 op. cit.), only partially blockedthe binding of [¹²⁵I]-HK to purified curli (1000 fold molar excessreduced the binding by 20-30%), suggesting that the binding site for HKis separate from those for fibronectin and plasminogen. The presence oflarge excess of human serum albumin finally, had no effect on thebinding of HK to curli (not shown).

Assembly of contact phase factors on curliated E. coli

HK is a major constitatent of the contact phase system of the humanplasma. Therefore we explored the possibility that other contactfactors, i.e. prekallikrein, factor XI and factor XII bind to E. coli.Furthermore we probed for the binding of LK, the low-molecular-weightsubstrate for kallikrein. Curli-expressing YMel bacteria bound the[¹²⁵I] labeled proteins at 37° C., whereas YMel-1 bacteria failed toshow significant binding (FIG. 12). As the control we used[¹²⁵I]-labeled radiolabeled human serum albumin which bound neither toYMel nor to YMel-1 bacteria. The results indicate that the entire set ofcontact phase factors, i.e. HK, prekallikrein, factors XI and XII, andthe kinin precursor LK, may assemble at the surface of curliatedbacteria

Bradykinin is released from HK bound to curliated E. coli

Plasma prekallikrein is activated by factor XII to α, kallikrein, andthe proteolytic action of α-kallikrein on HK or LK releases bradykinin.The finding that the various factors are assembled on E. coli promptedus to probe for the kinin present in HK eluted from the bacterialsurface. To this end we incubated E. coli bacteria with total humanplasma containing the entire set of contact factors. Using antibodyHKL16 to the light chain of HK we were able to detect a doublet band of114-120 kDa under non-reducing conditions (FIG. 13, right panel). Anidentical replica stained with a polyclonal antibody to bradykinin(A-BK) stained a single band of 120 kDa. Under reducing conditions (FIG.13, left panel) HKL16 detected three major bands, i.e. single-chain HKof 120 kDa, the intact light chain of 58 kDa, and a degraded form of thelight chain of 45 kDa (c.f. FIG. 2). Unlike HKL16, the α-BK antibodydecorated two major bands of 120 (single-chain HK) and 64 kDa (heavychain plus kinin segment) but none of the light chains. Notably (αkallikrein cleaves first at the carboxyterminal flanking site, Arg-Ser,of bradykinin, thereby separating the light chain from the heavy chainwhich still has bradykinin attached; in a second step kallikrein cleavesat the aminoterminal flanking site, Lys-Arg, thereby releasing thebradykinin peptide from the heavy chain. The cleavage of HK by plasmakallikrein to generate the nonapeptide bradykinin is highly specific(c.f. Silverberg and Kaplan 1988). Together our data indicate thatbinding of intact HK to the bacterial surface is followed by a (partial)cleavage of HK suggesting that bradykinin might be released from HKunder these conditions. It should be pointed out that the degradation ofthe HK light chain by α-kallikrein is often indicative of theconcomitant release of bradykinin from the heavy chain—The plasma sampleused for absorption was also incubated without adding bacteria. In thiscase no degradation of HK was observed (not shown).

To directly follow the release of immunoreactive bradykinin, curliatedYMel bacteria were preincubated with fresh human plasma, followed byextensive washing. We then applied α-kallikrein for various timeperiods, and eluted the surface-bound HK from E. coli. The resultantmixture was analyzed by Western blotting and immunoprinting using theanti-bradykinin antiserum (FIG. 14). After 5 min of incubation, almostall of the 120 kDa (single-chain HK) and most of the 64 kDa band (heavychain plus bradykinin) had disappeared. At 20 min almost all of thebradykinin immunoreactivity had disappeared, suggesting that bradykininhad been completely liberated. The results clearly indicate a rapid andefficient cleavage of HK under the release of immunoreactive bradykinin.

Discussion

The structural gene for the curli subunit is present in most naturalisolates of E. coli, but in vitro curli are expressed preferentially atgrowth temperatures below 37° C. (Olsen et al., 1989, op. cit). To whatextent curli are present on E. coli growing in vivo is not known, butthe fact that the level of expression varies considerably among clinicalisolates grown at 26° C. (see FIG. 2), indicates that this could be thecase also at 37° C. This notion is supported by the finding that somestrains of Salmonella express so called thin aggregative fimbriae(Collinson et al., 1991, op.cit.) also when grown at 37° C. (Mikael Relmand Staffan Normark, personal communication). These surface fimbriae areclosely related to curli (Collinson et al., 1991, op. cit.; Olsen etal., 1993, op cit). It could also be that curli are presentpredominantly when E. coli primarily infects and colonizes the humanhost, or that curli are expressed in vivo under specific environmentalconditions. Finally, also a low degree of curli expression could beenough to reach local concentrations of HK and other contact phasefactors sufficient to elicit blood coagulation and/or bradykininrelease.

Previous work has demonstrated that curli interact with components ofthe extracellular matrix and the fibrinolytic system (Olsen et al op.cit, 1989; Sjobring et al, 1994, op. cit). The starting point for thepresent experiment was the binding of HK to various bacterial species,and the subsequent finding that HK in the case of E. coli, binds tocurli. An important question raised by the interactions between curliand different host proteins concerns the specificity of a giveninteraction. Does for instance the binding of HK occur in vivo in thepresence of other curli-binding proteins? The fact that M likefibronectin and plasminogen (Olsen et al., 1989, op. cit; Sjobring etal., 1994, op. cit), is bound to curliated E. coli in plasma environmentshows that this is the case.

The ability of curli to interact with a variety of host proteins providethe bacteria with adhesive, invasive, and virulence properties. In afirst step curli may mediate adherence to cellular and matrix components(Olsen et al, 1989, op. cit). As the infection progresses, theinteraction with plasminogen and tissue-type plasminogen activator (tPA)could promote the spreading of the infection by degradation of tissuesvia activated plasmin (see Parkkinen, L, Hacker, L, and Korhonen, T. &(1991) Enhancement of tissue plasminogen activator catalyzed plasminogenactivation by Escherichia coli S fimbriae associated with neonatalsepticaemia and meningitis. Thromb Haemost 65: 483-486.; Lottenberg, R-,Minnin-Wenz, D., and Boyl, M. D. P. (1994) Capturing host plasmin(ogen):a common mechanism for invasive pathogens? Trends Microbiol 2: 20-24;Lahteennmaki K., Virkola, R., Pouttu, R-, Kuusela, P., Kukkonen, M., andKorhonen, T. K. (1995) Bacterial plasminogen receptors: in vitroevidence for a role in degradation of the mammalian extracellularmatrix. Infect Immun 63: 3659-3664). In the case of HK the bacteriamight exploit the permeability-increasing properties of the cognateeffector peptide, bradykinin, in the infectious process. The observationthat not only HK, but also other components of the contact phase systembind to curli, suggests that curliated E. coli could act as platformsfor the assembly of such components leading to a subsequent activationof the procoagulatory and the proinflammatory pathways. The notion thatbacterial endotoxins are potent activators of factor XII (Morrison,D.C., and Cochrane, C. G. (1974) Direct evidence for Hageman factor(factor XII) activation by bacterial lipopolysaccharides (endotoxins). JExp Med 140: 797-811) which in turn activates prekallikrein and viceversa (FIG. 1) lend further support to our hypothesis. In this waybacteria loaded with the elaborate contact phase activation system mightpromote the excessive bradykinin release that is not controlled by theotherwise tightly regulated mechanisms of homeostasis. In the case ofsepticemia a release of bradykinin might produce vasodilation andincrease vascular permeability, effects which cause the leakage ofplasma, hypovolemic hypotension, and, in severe cases, circulatoryshock. Furthermore, the initiation of the intrinsic blood coagulationcascade via surface-bound factor XIa might contribute tohypercoagulability and even to disseminated intravascular coagulation,which represents a serious complication to sepsis. Finally, theacquisition of a surrounding clot could protect the bacteria from thehost defense mechanisms.

Microbial pathogenicity and virulence are highly polygenic properties,and the complexity and multitude of molecular interactions creating thehost-microbe relationship makes it difficult to predict the effect(s) ofa certain interaction. However, if disturbances in the balance betweenan infecting microorganism and its host causing disease are to becorrected therapeutically, it is necessary to define the molecularinterplay which represents the basis for the relationship. The presentexample describes potential virulence mechanisms which may be used astherapeutic targets, while underlining the complexity of thehost-microbe relationship.

Thus, the present invention provides the necessary basis for creatingtherapeutic agents effective against a wide variety of conditions. Oneexample is Systemic Inflammatory Response Syndrome, previously known assepsis, septic shock, sepsis syndrome etc, which is caused byinflammatory responses to a variety of stimuli, of which some areinfectious in origin (see E. T. Whalley and J. C. Cheronis). Accordingto the present invention, therapeutic agents may be provided, whichincrease the chances of survival in patients suffering from e.g. thissyndrome, even though the underlying etiology is still unknown.

Example 3

Bacterial strains—S. pyogenes strains AP 1 (40/58) and AP74 (30/50) arefrom the World Health Organisation Collaborating Centre for Referencesand Research on Streptococci Institute of Hygiene and Epidemiology,Prague, Czech Republic.

Sources of proteins and antibodies—H-kininogen was isolated from humanplasma (Salvesen, G., C. Parkes, M. Abrahamson, A. Grubb, and A. J.Barrett. 1986. Human low-Mr kininogen contains three copies of acystatin sequence that are divergent in structure and in inhibitoryactivity for cysteine proteinases. Biochem. J 234:429-434 withmodifications previously described (Hasan, A. A., D. B. Cines, J. Zhang,and A. H. Schmaier. 1994. The carboxyl terminus of bradykinin and aminoterminus of the light chain of kininogens comprise an endothelial cellbinding domain—J. Biol. Chem. 269:31822-31830). The streptococcalcysteine proteinase (SCP) was purified from the culture medium of strainAP1 (Berge, A., and L. Bjorck. 1995. Streptococcal cysteine proteinasereleases biologically active fragments of streptococcal surfaceproteins. J Biol. Chem. 270:9862-9867. The AP1 supernatant was subjectedto ammonium sulfate precipitation (80%) followed by fractionation onS-Sepharose in a buffer gradient (5-250 mM MES, pH 6.0). The zymogen wasfurther purified by gel filtration on Sephadex G-200. Monoclonalantibodies to human kininogens (HKH 15 and HKL, 9) were produced in mice(Kaufmann, J., M. Haasemann, S. Modrow, and W. Muller-Esterl. 1993.Structural dissection of the multidomain kininogens. Fine mapping of thetarget epitopes of antibodies interfering with their functionalproperties. J Biol. Chem. 268:9079-9091.), polyclonal antiserum (AS88)to human H-kininogen in sheep (Muller-Esterl, W., D. Johnson, G.Salvesen, and A. A. Barrett. 1988. Human kininogens. Methods Enzymol.163:240-256), and polyclonal antiserum against the streptococcalcysteine proteinase were raised in rabbits. Antiserum to bradykinin ((xBK, AS348) was produced in a rabbit by previous coupling of the cognatepeptides to keyhole limpet hemocyanin (KLH) via the carbodiimide method(Herwald, H., A. H. K. Hasan, J. Godovac-Zimmermann, A. H. Schmaier, andW. Muller-Esterl. 1995. Identification of an endothelial cell bindingsite on kininogen domain D3. J Biol. Chem. 270:14634-14642).Peroxidase-conjugated goat antirabbit goat anti-mouse (Bio-Rad,Richmond, Calif.), or donkey anti-sheep immunoglobumin (ICN, Aurora,Ohio) were used as secondary antibodies. The Z-Leu-Val-Gly-CHN₂ peptidehas been described (Bjorck, L., P. Akesson, M. Bohus, J. Trojnar, M.Abraham I. Olafsson and A. Grubb. 1989. Bacterial growth blocked by asynthetic peptide based on the structure of a human proteinaseinhibitor. Nature 337:385-386).

Cleavage off H-kininogen by SCP—H-kininogen (0.5 mg/ml) was incubated at37° C. with SCP in 10 mM NaH₂PO₄, 10 mM Na₂HPO₄, 0.15 M NaCl, pH 7.4(PBS) containing 1 mM dithiothreitol (DTT); the molar ratio of substrateover enzyme was 100:1 or 1:1. Aliquots (8 μl) of the reaction mixturewere removed at the indicated time points, and the reaction stopped byadding 10 μl of a 2% (w/v) sodium dodecyl sulfate (SDS) sample buller(Laemmli, U.K. 1970. Cleavage of structural proteins during the assemblyof the head of bacteriophage T4. Nature 227:680-685) containing 5% (v/v)2-mercaptoethanol, and boiling at 95° C. Alternatively the reaction wasstopped by addition of 10 μM (final concentration) of [N-(L-3transcarboxyoxiran-2-carbonyl)-L-leucyl]-agmatin (E-64).

Cleavage of plasma prekallikrein by SCP—Plasma prekallikrein (16 μg) wasincubated with 0.05-0.5 μg of SCP in 100 μl of PBS containing 1 mM DTTat 37° C. for 60 min; the molar ratio was 10:1 to 100:1. The reactionwas stopped by adding 10 μl of SDS sample buffer containing 5%2-mercaptoethanol and boiling at 95° C.; alternatively E-64 was added toa final concentration of 10 μM.

Prekallikrein activation—Plasma prekallikrein (4 μg) was incubated for 1h with 0.012 μg factor XIIa in 40 μl of PBS, or for 3 h with varyingamounts (0.12 to 0.012 μg) of SCP at 37° C. To test the activity of thegenerated proteinase, kallikrein was added to 200 μl of a 0.6 mMsolution of S-2302 (H-D-Pro-Phe-Arg-p-nitro-anilide, Haemochrom,Diagnostica, Essen, Germany) in 0.15 M Tris-HCl, pH 8.3. The substratehydrolysis was measured at 405 nm.

Cleavage of plasma proteins by SCP—One hundred μl of human plasma wasincubated with 3.2 μg of SCP dissolved in 100 μl PBS, 10 mM DTT, pH 7.4,at 37° C. The reaction was stopped by the addition of 100 μl of SDSsample buffer containing 5% 2-mercaptoethanol and boiling at 95° C. for5 min.

SDS-polyacrylamide gel electrophoresis (PAGE)—Proteins were separated by10 or 12.5% (w/v) polyacrylamide gel electrophoresis in the presence of1% (w/v) SDS Standard molecular weight markers were from Sigma.

Western blotting and immunoprinting—Proteins were resolved by SDS-PAGEand transferred onto nitrocellulose membranes for 30 min at 100 mA(Khyse-Andersen, J.1984. Electroblotting of multiple gels: a simpleapparatus without buffer tank for rapid transfer of proteins frompolyacrylamide to nitrocellulose. J. Biochem. Biophys. Methods10:203-209) The membranes were blocked with 50 mM KH₂PO₄, 0.2 M NaCl, pH7.4, containing 5% (w/v) dry milk powder and 0.05% (w/v) Tween 20.Immunoprinting of the transferred proteins was done according to Towbin,H, T. Staehelin, and J. Gordon. (1979. Electrophoretic transfer ofproteins from polyacrylamide gels to nitrocellulose sheets: procedureand some applications. Proc. Natl. Acad Sci. USA 76:4350-4354). Thefirst antibody was diluted 1:1000 in the blocking buffer (see above).Bound antibody was detected by a peroxidase-conjugated secondaryantibody against sheep, rabbit or mouse immunoglobulin followed by thechemiluminescence detection method.

Ca2⁺ release from intracellular stores—Human foreskin fibroblasts(HF-15) on 10 mm diameter glass coverslips were grown to confluency inDulbecco's modified Eagle's medium supplemented with 10% (v/v) fetalcalf serum (Quitterer, U., C Schroder, W. Muller-Esterl, and H.Rehm—1995. Effects of bradykinin and endothelinl on the calciumhomeostasis of mammalian cells. J Biol. Chem. 270:1992-1999). The cellswere washed twice with minimum essential medium buffered with 20 mMNa+-HEPES, pH 7.4 (buffer A; without vitamins, and α-D-glucose addedimmediately before use). The cells were loaded for 30 min at 37° C. with2 μM1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2′-amino-5′-methylphenoxy)-ethane-N,N,N′,N′-tetra acetic acid, pentaacetoxymethylester(fura2/AM; Calbiochem, San Diego, Calif.) in buffer A containing 0.04%(w/v) of the nonionic detergent pluronic F-127 (Calbiochenl, San Diego,Calif.) (see above). The cells were washed twice with buffer A. TheHitachi F4500 fluorescence photometer was employed with the excitationwavelength alternating between 340 mn and 380 nM and the emissionwavelength set at 510 nm. To induce the Ca²⁺ release, 2 μg H-kininogenor proteolytic cleavage products thereof in 20 μl of reaction buffer wasadded 60 s after starting the measurement. The release of Ca²⁺ fromintracellular stores was followed for 300 s; the free intercellular Ca²⁺concentration was calculated from the ratio of 340 nm/380 nm asdescribed (ses above).

Determination of kinin concentrations in plasma—To measure theSCP-induced kinin release 100 μl of plasma was incubated with 3.2 μg ofSCP in 100 μl PBS containing 10 mM DTT. Samples (10 μl each) wereremoved after 0, 30, 60, 90, and 120 min. The reaction was stopped byadding E-64 to a final concentration of 10 μM. For control 100 μl ofhuman plasma was incubated with buffer in the absence of SCP. Thesamples were diluted 1:100 in distilled water. Aliquots (100 μl each)were mixed with 20 μl of 20% (w/v) trichloroacetic acid and centrifugedat 1500×g for 10 min. The kinin concentrations in the reaction mixtureswere quantitated by the Markit-A kit (Dainippon Pharmaceutical Co.,Osaka, Japan) as described (Scott, C. F., E. J. Whitaker, B. F. Hammond,and R-W. Colman. 1993. Purification and characterization of a potent70-kDa thio lysyl-proteinase (Lys-gingivain) from Porphyromonasgingivalis that cleaves kininogens and fibrinogen. J Biol. Chem.268:7935-7942). Briefly, aliquots of the supernatant (75 μl each) weremixed with 75 μl of the kit buffer, and applied to the wells (100 μleach) of microtiter plates that were coated with capture antibodies torabbit immunoglobulin, followed by specific anti-bradykinin antibodies.After 1 h of incubation, the peroxidase-labelled bradykinin probe wasapplied and incubated for 1 h. The amount of bound peroxidase wasvisualised by the substrate solution, 0.1% (w/v)diammonium-2,2′-azino-bis-(3-ethyl-2,3-dihydrobenzthiazoline)-6-sulfonate(ABTS), 0.012% (v/v) H₂O₂ in 100 mM citric acid, 100 mM NaH₂PO₄, pH 4.5for 30 min. The change of absorbance was read at 405 nm. The referencestandards were prepared according to the manufacture's instructions.

Animal experiments—S. pyogenes of strains API and AP74 were grown inTodd Hewitt broth (Difco, Detroit Mich.) at 37° C. for 16 h, andharvested by centrifugation at 3000×g for 20 min. The bacteria werewashed twice with PBS, and resuspended in PBS to 3×10⁸ cells/ml. One mlof living bacteria was injected intraperitoneally into outbred NMRImice. Plasma samples were taken 10 h after injection. Alternatively,mice were injected with the purified non-activated SCP (0.1 to 0.5 mg),and plasma samples were taken 60 min, 150 min, and 300 min afterinjection. For inactivation of SCP, 0.5 mg of the enzyme was mixed with0.2 mg Z-Leu-Val-Gly-CHN₂ prior to injection. To monitor the cleavage ofkininogen, 1 μl of plasma was run on SDS-PAGE followed by Westernblotting with antibodies against bradykinin (α-BK), Quantification ofSCP in mouse plasma—One μl of plasma samples from mice injected with SCPwas run on SDS-PAGE and transferred onto nitrocellulose. The enzyme wasvisualized by immunostaining using antibodies against SCP. To obtainsemi-quantitative estimates of the SCP amounts in plasma samples,purified SCP (3 ng to 100 ng) was processed as described above and usedas a standard.

Results

Streptococcal cysteine proteinase is not inhibited by H-kininogen. Thestreptococcal cysteine proteinase (SCP) cleaves surfaces proteins of S.pyogenes strain AP 1 (see above). One of its target structures, thestreptococcal M 1 protein, specifically binds kininogens, (Ben Nasr, A.B., H. Herwald, W. Muller-Esterl, and L. Bjorck. 1995. Human kininogensinteract with M protein, a bacterial surface protein and virulencedeterminant. Biochem. J 305:173-180.) the major cycsteine proteinaseinhibitors of human plasma. These observations prompted the notion thatkininogens bound to the bacterial surface might regulate the proteolyticactivity of SCP. We therefore tested the effect of H-kininogen on thehydrolysis of a chromogenic peptide substrate by SCP. Unexpectedly,H-kininogen had no inhibitory effect on the amidolytic activity of SCP(not shown), whereas the synthetic cysteine proteinase inhibitor E-64,efficiently blocked the SCP activity in the same assay. We thereforeasked the question whether H-kininogen serves as a substrate—rather thanan inhibitor for SCP.

SCP degrades H-kininogen. When we analyzed the reaction mixture ofH-kininogen and SCP by SDS-PAGE we found that SCP rapidly and almostcompletely degraded H-kininogen (FIG. 15). To follow the breakdown ofH-kininogen by SCP, and to identify potential cleavage products such asthe biologically active kinin peptides, we employed Western blotting andimmunoprinting of the reaction mixtures. We used polyclonal antibodiesdirected to the kinin sequence of 9 residues located in domain D4 ofH-kininogen, and monoclonal antibodies to the flanking domains, D3 andD5H (of FIG. 3). FIG. 16 shows three replicas of the SCP cleavageproducts of H-kininogen separated by SDS-PAGE and immunoprinted by amonoclonal antibody against the COOH-terminal part of domain D3 (HKH 15;FIG. 16A), by a polyclonal antibody to bradykinin (α-BK; FIG. 16B), andby a monoclonal antibody recognizing the NH₂-terminal part of domain D5H(HKL 9; FIG. 16C), respectively. The imimmoprints reveal a complexpattern of kininogen degradation products. The native H-kininogen of 105kDa is rapidly cleaved into fragments of 60 to 75 kDa containing the D3epitope (panel A), and into fragments of 45 to 70 kDa comprising the D5Hepitope (C). Initially the kinin epitope which is rapidly lost from thenative kininogen of 105 kDa, remains associated with a band of 60 kDathat is also recognized by the anti-heavy chain antibody (A) but not bythe anti-light chain antibody (C). This would indicate that the initialcleavage by SCP occurs at site(s) located distally of the bradykininmoiety, and therefore the bradykinin sequence remains attached to theheavy chain. Further proteolysis by SCP breaks down the kininogen heavychain, most probably into its constituting domains (note that thevarious domains D1 through D3 of the kininogen heavy chain are separatedby protease-sensitive regions that expose the primary attack sites formany proteinases; (Vogel, R-, I. Assfalg Machleidt A. Esterl, W.Machleidt and W. Muller-Esterl. 1988. Proteinase-sensitive regions inthe heavy chain of low molecular weight kininogen map to theinter-domain junctions. J Biol. Chem. 263:12661-12668.). Accordingly, aprominent band of approximately 23 kDa appears at the later stages ofproteolysis representing domain D3 (B). A fraction of D3 still containsthe COOH-terminal extension of bradykinin (B, lanes 3 and 4) that islost as proteolysis proceeds (120 min). No shift in the apparentmolecular mass of the D3 fragment is obvious (see A, lanes 3 to 6)suggesting that only a minor peptide such as bradykinin is removed fromthe 23 kDa fragment. Nevertheless, we seeked to determine whether SCPreleases authentic kinins from H-kininogen.

H-kininogen cleavage products release intracellular Ca2+ inhumanfibroblasts. To demonstrate the presence of biologically activekinins in the proteolytic digests we employed the fura-2/AM assay. Thistest system monitors the bradykinin B2 receptor mediated release of Ca²⁺from intracellular stores of human foreskin fibroblasts (see above).Purified H-kininogen did not induce a Ca²⁺ release from humanfibroblasts 41 (FIG. 17A.); hence the starting product did not containappreciable amounts of kinins. In contrast the reactions mixtures fromthe incubation of H-kininogen with SCP for 60 min (C) or 120 min (D)induced significant Ca²⁺ signals thus indicating the presence ofbiologically active kinins. The specificity of the assay was probed bypreincubating the cells with the potent B2 receptor antagonist HOE 140,which completely abrogated the Ca²⁺ signal induced by the application ofthe kininogen breakdown products (data not shown). H-kininogen which hadbeen incubated for 120 min in the absence of SCP induced no Ca²⁺ signal(data not shown); hence the kinin release was not due to a contaminatingkininogenase associated with the starting material. Together theseresults demonstrate that SCP releases biologically active kinins fromH-kininogen.

SCP cleaves H-kininogen in plasma. To test whether SCP cleavesH-kininogen in its physiological environment the streptococcal enzymewas added to plasma. After varying time points, aliquots were removedfrom the reaction mixture and subjected to Western blot analyses. Highlyspecific polyclonal antibodies to native H-kininogen (AS 88) and tobradykinin (α-BK) were applied to identify the kininogen cleavageproducts in the complex plasma mixture. FIG. 1 SA demonstrates that theendogenous H-kininogen present in human plasma is partially degradedafter 15 min, and almost completely split after 30 min of incubationwith SCP. Because the antiserum. (AS88) is primarily directed toimmunodominant epitopes of the H-kininogen light chain (20) it poorlycrossreacts with L-kininogen which is seen as a faint band of 66 kDa (A,lane 1; cf. B, lane 1). The α-BK antibodies reacted weakly with thenative forms of H-kininogen and L-kininogen, respectively (B, lane 1).After 15 min of incubation a strong immunoreactivity at 66 kDa isvisible which likely corresponds to a kinin-containing fragmentrepresenting the kininogen heavy chain including the bradykinin epitope(note that the cleavage of a scissile bond flanking the kinin segmentresults in a major conformational change of the kininogen molecule and aconcomitant exposure of the bradykinin epitope). Under the conditions ofour experiment SDS-PAGE does not resolve the putative fragment andL-kininogen because the proteins dilfer only by 36 residues. After 15min of SCP proteolysis an smaller fragment of approximately 60 kDa isrecognized by the anti-bradykinin antibodies (B, lane 2). This latterfragments which peaks at 30 min (B, lane 3) and fades away afterprolonged incubation is likely to represent a degradation product of thekinfflogen heavy chain with bradykinin still attached to itscarboxyterminus. Unlike the former band, i.e. heavy chain comprising thebradykinin epitope, the latter band, presenting a putative heavy chaindegradation product is not observed when kininogen is split by itsphysiological processing enzyme, plasma kallikrein (see above). Theprominent 45 kDa band which occurs throughout the entire incubationprocedure (FIG. 18B) is likely to be a staining artefact of the (α-BKantibodies when plasma is used; we did not observe such animmunoreactivity with purified H-kininogen (see FIG. 16). We could notdetect any significant kininogen degradation in plasma that wasincubated in the absence of SCP (data not shown). Together these datasuggest that SCP degrades kininogen both in an isolated system and incomplex mixtures such as plasma, and that the rapid loss of kininimmunoreactivity reflects the liberation of the hormone by SCP.

SCP does not activate purified plasma prekallikrein. Under physiologicalconditions, the kinin release from kininogens is mediated by activatedplasma kallilcrein. Due to a reciprocal activation factor XIII convertsthe zymogen, prekallikrein, to the active enzyme, α-kallikrein. Hence,activation of plasma prekallikrein by SCP may explain at least in partthe observed release of kinins, from H-kininogen (see above). To testthis possibility, prekallikrein isolated from human plasma was incubatedwith purified SCP, and followed by SDS-PAGE demonstrating thatprekaffihein was rapidly processed by the streptococcal enzyme (data notshown). The resultant cleavage products were tested for their amidolyticactivity in chromogenic assays using the pnitroanilide derivative of thetripeptide, H-D-Pro-Phe-Arg. Prekallikrein cleavage products generatedby varying concentrations of SCP did not reveal significant amidolyticactivity (FIG. 19); likewise prekallikrein or SCP alone had no activity.In contrast prekallikrein activation by factor XIIIa resulted in theprogressive activation of the zymogen. Because SCP is unable to activateprekallikrein under the conditions of our experiment we conclude thatthe bacterial enzyme is likely to act directly on kininogen present inhuman plasma without prior activation of a physiological kininogenase.This notion is supported by the observation that kininogen degradationproducts are formed by SCP that do not occur in the krein-mediatedprocessing cascase.

SCP generates kinins from plasma kininogens. Our proteolysis experimentsdemonstrated that biologically active kinins are released fromH-kininogen by SCP in a purified system. We therefore asked the questionwhether SCP may liberate kinins from kininogens also in a complexenvironment such as the plasma. To this end we incubated human plasmawith purified SCP for 2 h and tested aliquots of the reaction mixtureafter varying time periods. A competitive ELISA was employed and FIG. 20demonstrates that SCP release kinins in a time-dependent manner. After120 min of incubation, the kinin concentration of samples had levelledoll at 2.8 μM which almost approaches the theoretically releasableconcentration of bradykinin in human plasma of 3.5 μM. Thus,approximately 0.9 μM H-kininogen and 2.6 μM L-kininogen are present inhuman plasma (Mfiller-Esterl, W. 1987. Novel functions of kininogens.Sem. Thromb. Hemostas. 13:115-126). No release of kinins was found incontrols where plasma was incubated without SCP. These resultsdemonstrate that SCP-induced cleavage of kininogen in plasma is combinedwith the release of kinin.

SCP cleaves kininogens in vivo. To test whether SCP also processeskininogens in vivo, we injected purified SCP into the peritoneal cavityof mice. Two types of experiments were performed. In the first set ofexperiments, lethal doses of SCP (0.5 mg per animal) were administratedintraperitoneally, and plasma samples from these animals were taken 60min, 150 min, and 300 min after injection (FIG. 21A). For control, 0.5mg SCP that had been inactivated by the specific inhibitor Z-Leu-Val-GlyCHN₂ (See above) was injected i.p. into mice, and plasma samples werewithdrawn after 300 min. In a second set of experiments, varying amountsof SCP (0.1-0.5 mg) were injected i.p., and plasma samples were taken300 min thereafter (FIG. 21B). Kininogen degradation in plasma wasdetected by Western blotting, using antibodies to bradykinin. Threeimmunoreactive band of 66, 80, and 110 kDa were detected in plasma ofmice that had been treated with vehicle only; the upper 110 kDa band and44 the lower 66 kDa band correspond to H- and L-kininogen, respectively.The intermediate band of 80 kDa may correspond to a modified form ofmouse Lkininogen, ir-kininogen, that has recently been described inmouse fibroblasts (Takano, M., K. Yokoyama, K. Yayarna, and H. Okamoto.1995. Murine fibroblasts synthesize and secrete kininogen in response tocyclic-AMP, prostaglandin E2 and tumor necrosis factor. Biochim.Biophys. Acta 1265:189-195). Plasma of mice that had been injected withSCP 60 min prior to bleeding completely lacked the immunoreactiveH-kininogen band of 110 kDa. After 150 min most of the plasma kininogenshad been degraded, and after 300 min no kinning fragments weredetectable. By contrast the majority of plasma kininogens from animalsthat had been injected with the enzyme-inhibitor complex remainedintact.

A dose-dependent effect of SCP on plasma kininogen degradation was foundwhen we injected increasing amounts of SCP (FIG. 21B). Even at lowestenzyme amounts (0.1 and 0.2 mg) a significant fraction of plasmakininogens was found to be degraded. At high SCP amounts 0.4 mg) hardlyany kinin-containing kininogen fragments or fragments thereof weredetectable. From semi-quantitative Western blot analyses we judged theplasma concentration of SCP to be in the range of 3-25 μg/ml of plasmadependent on the amount of injected enzyme and the time elapsed afterinjection (Table 2).

Alternatively, living streptococci of strain AP 1 were injected i.p..Plasma samples were drawn then from the animals 8 h after injection, andanalyzed by SDS-PAGE). (FIG. 22). Coomassie Brilliant Blue stainingshowed no apparent difference between normal mouse plasma and plasmasamples from mice injected with SCP or AP 1 bacteria demonstrating thatthe overall protein composition of plasma was unchanged (FIG. 22A). Inthe corresponding Western blots, SCP was detected in the plasma of micegiven 0.5 mg of the enzyme i.p., but not in the plasma of mice infectedwith AP 1 bacteria, indicating that the concentration of SCP was lowerin the latter experimental setting (data not shown). This observationmay also explain why kininogens were not completely degraded in theseanimals (see FIG. 21B). Immunoprinting of the plasma samples with α-BKantibodies revealed that native H-kininogen was completely absent fromthe plasma of mice treated with SCP as evidenced by the kininimmunoreactivity. Furthermore, kininogen concentrations wereconsiderably though not completely reduced in the plasma of miceinfected with S. pyogenes of the AP 1 strain (FIG. 22B). These findingsdemonstrate that kininogens are also degraded by SCP in vivo, mostlikely under the release of kinins. For control we used AP 74 bacteria,the only strain of S. pyogenes that we have found not to produce SCP(Cooney. Liu, and Bjorck, manuscript in preparation). No significantdecrease of kininogens was seen in plasma of mice treated with the sameprotocol as above except that AP 74 bacteria were used (not shown), thusunderlining the specific role for SCP in kininogen turnover and kininrelease. Together, these data demonstrate that purified SCP or SCPsecreted by S. pyogenes, cleaves kininogens in vivo under the release ofkinins.

TABLE 2 Quantification of SCP in Mouse Plasma Amount of SCOP Time of thePlasma concentration administered (i.p.) (mg) administration (min)(μg/ml) 0.5  60 12 0.5 150 20 0.5 300 25 0.1 300  2 0.2 300 12 0.3 30012 0.4 300 20

Example 4

The effects of bradykinin antagonists during bacterial infections werestudied in vivousing the Streptococcus pyogenes strain AP 1 (Akesson etal., Mol. Immunol., vol 27pp. 523-531) and one of its virulence factors,the streptococcal cysteine proteinase (SCP). In different sets ofexperiments the bradykinin antagonist HOE 140 (Bao et al. (1991), supra)was injected intraperitoneally or subcutaneously into outbread Nnflumice. The experiments were designed as follows; mice were injected withlethal doses of SCP intraperitoneally (0.5 mg/animal) or living AP 1bacteria subcutaneously in an air pocket (10⁷ bacteria/animal). 5 hoursafter injecting SCP the mice which received HOE 140 still lookedhealthy, whereas the mice which did not receive any bradykininantagonist died. 35 hours after the AP 1 injection the mice who receivedHOE 140 appeared to be healthy, whereas those who did not appeared to bevery ill.

Example 5 Activation of the Contact Phase System on E. coli andSalmonella

Bacterial strains and culture conditions. YMel is a curli-expressing E.coli K12 strain. YMel-1 is an isogenic curli-deficient mutant straingenerated by insertional mutagenesis of the csgA gene in YMel. SR11B isa S. typhimurium SR-11 X4666 strain; csgA and csgB mutant strains (SR-11X4666, csgA::Tn1731; SR-11 X4666, csgB::Tn1721) were produced bytransposon mutagenesis. Bacteria were grown on colonization factorantigen agar at 37° C. (S. typhimurium) or at 26° C. (E. coli) andbacteria were washed and resuspended in 50 mM HEPES, 2 mM CaCl₂, 50 μMZnCl₂, pH 7.35 containing 0.02% (w/v) NaN₃ and 0.05% (v/v) Tween 20(buffer A). Sources of proteins, HK and PK were isolated from humanplasma. F XI and F XII were from Enzyme Research Laboratories (Swansea,UK). Proteins were [¹²⁵] iodinated by the chloramine-T method of Frakerand Speck supra.

Bacterial binding assay. Harvested bacteria were suspended in buffer Ato a final count of 2×10⁸ to 2×10¹⁰ cell per ml. The bacterialsuspension (200 μl) was incubated with radioactively labeled protein atroom temperature for 10 to 120 min. Following addition of 2 ml ofbuffer, the cell suspension was centrifuged, and the radioactivity ofthe pellet was counted. Binding activity is given as the percentage ofbound vs total radioactivity per tube. For electrophoretic analysis, thebacterial pellet was resuspended in 20 μl reducing SDS sample buffer.The samples were boiled at 95° C. followed by centrifugation (3000 g, 5min) and SDS-PAGE. The mixture of 4 contact-phase factors included0.2×10⁻¹² M F XI, 3.0×10⁻¹² M F XII, 3.0×10⁻¹² M PK and 4.6×10⁻¹² M HK(final concentrations). For autoradiography gels were dried and exposedto Kodak Scientific Imaging Film for 1 to 4 days (Eastman Kodak,Rochester, N.Y.).

Bradykinin determination. Human plasma samples (0.5 ml each ) wereincubated with 0.5 ml bacteria (2×10¹⁰ bacteria/ml). Followingincubation the bacteria were recovered and washed as detailed above.Pellets were resuspended in 0.5 ml buffer A. After a 15 min incubationat room temperature, the bacteria were centrifuged at 3000 g for 5 min.The supernatants were acidified with an equal volume of 0.1% (v/v)trifluoroacetuc acid, and applied to a SEP-Pak Light C18 cartridge(Waters Corp., Milford, Mass.) pre-equilibrated with the same buffer.The column was washed with 6.0 ml 60% (v/v) acetonitrile in 0.1%trifluoroacetic acid. Fractions were lyophilized and redissolved in 250μl RIA buffer (Bradykinin radioimmunoassay kit; Peninsula Laboratories,Belmont, Calif.), and their bradykinin concentration was determinedfollowing the manufacturer's instructions.

Clotting assay. The clotting time was measured in a coagulometer(Amelung, Lemgo, Germany). A bacterial suspension (6×10⁴ cells/30 μl of50 mM HEPES, 50 μM ZnCl₂, pH 7.35) was incubated with 100 μl of sodiumcitrate-treated normal human plasma for 30 s, followed by incubationwith Platelin LS (aPTT kit; Organon Teknica, Durham, N.C.) orphospholipids only (37.5% phosphatidyl choline, 37.5% phosphatidylethanolamino and 35% phosphatidyl serine, all were obtained from SigmaChemical (St. Louis, Mo.). To initiate clotting 100 μl of 25 mM CaCl₂was added.

Scanning electron microscopy. Clots were gently drawn down onto aMillipore filter (Waters Corp) by suction caused by a wet filter paperlying underneath. The filters were fixed in 2% (v/v) glutaraldehyde, 0.1M cacodylate, 0.1 M sucrose, pH 7.2 for 1 h at 4° C., and washed with0.15 M cacodylate, pH 7.2. The filters were postfixed for 1 h with 1%(w/v) osmium tetroxide, 0.15 M sodium cacodylate, pH 7.2 for 1 h at 4°C., washed and stored in cacodylate buffer. Fixed filter paper sampleswere dehydrated with an ascending ethanol series (10 min per each step),dried, mounted on aluminium holders, sputtered with palladium/gold andexamined in a Jeol JSM-350 scanning electron microscope (Jeol, Chicago,Ill.).

Plasma protein concentrations following absorption with bacteria.Bacteria (2×10¹⁰/ml) in 0.11 M sodium citrate, pH 7.35, were incubatedfor 15 min with an equal volume of sodium citrate-treated human plasma,followed by centrifugation 5 min at 3000 g. F XI and F XII (human andmouse) were both measured with clotting-based assays using the aPTTreaction (activated partial thromboplastin time) and human deficiencyplasma. Protein C was measured with Coatest protein C kit formChromogenix (Protac-Agkistrodon Contortrix) and AS-2366 (Chromogenix AB,Molndal, Sweden). The concentration of remaining plasma proteins in thesupernatant were determined. Mouse fibrinogen was determined by amodified Schneider test.

Animal experiments. Female outbred NMRI mice were given an intravenousinjection of 0.5 ml of vehicle or bacterial suspensions (2×10¹⁰cells/ml) in 0.1 M phosphate, 0.15 M NaCl, pH 7.4; three animals wereused in each group. Blood was collected 15 min or 60 min after injectionby intracardiac puncture.

Binding of contact-phase proteins to curliated bacteria. Initially wetested the binding of the human contact-phase proteins F XI, F XII, PKand HK to curliated Escherichia coli (strain Ymel) and Salmonellatyphimurium (strain SR11B) bacteria. As a control, one isogenic E. colimutant strain (strain Ymel-1) and two S. typhimurium mutant strains(strains CsgA and CsgB), all lacking curli expression were used.Curliated bacteria, but not curli-deficient mutant strains, avidly boundthe various radiolabelled contact factors (FIG. 23) demonstrating thatthe curli proteins play a crucial role in contact factor binding.

Limited proteolysis of the contact factors indicates the activation ofthe corresponding pathway. Radiolabelled contact phase factors that hadbeen isolated from human plasma were individually incubated withcurliated bacteria for 10 to 45 min. Samples were drawn, and theabsorbed proteins were eluted from bacteria and analysed by SDSpolyacrylamide gel electrophoresis (SDS-PAGE) followed byautoradiography. Upon incubation with bacteria, F XII was readilycleaved into two major products of 30 and 50 kDa (FIG. 24A) which likelycorrespond to the heavy and light chain of F XIIa respectively. Incontrast HK, PK and F XI remained intact (data not shown).

The interaction of contact factors with bacterial surfaces was furtheranalyzed by incubating curliated strains SR11B and Ymel with a mixtureof the four contact factors that reflected their molar ratios in humanplasma. Only one factor was radiolabeled in each experiment. Incubationof ¹²⁵I-labeled PK in the presence but not in the absence of F XI, F XIIand HK resulted in the typical cleavage pattern indicative of PKactivation (FIG. 24B). Likewise ¹²⁵I-labeled HK was cleaved into itsheavy and light chains of 65 and 66 kDa, respectively (FIG. 24C) whenthe mixture of contact factors was present, suggesting that kinin wasreleased. In contrast, no such activation occurred when the mixture ofcontact factors was incubated in the absence of bacteria (data notshown). These results that the contact-phase system is activated uponexposure to bacteria surfaces and that the initial step is likely to beF XII activation.

Interactions in plasma environment.

The curliated Ymel and SR11B strains were incubated with whole humanplasma; the curli-defiant isogenic mutant strains YMel-1, CsgA and CsgBserved as the controls. Proteins bound to the bacteria were solubilized,separated and subjected to Western blot analysis. F XI, F XII, PK and HKwere absorbed from plasma by the curliated bacteria, but not by themutant strains (data not shown). Furthermore, proteolytic activity of FXIa, F XIIa and PKa was associated with Ymel and SR11B bacteria, asdemonstrated by chromogenic substrate assays; no such activity wasdetectable in noncurliated strains (data not shown). To monitor theinitiation of the contact-phase system on bacterial surfaces, weexamined the release of bradykinin by radioimmunoassay followingincubation of plasma for 10 minutes with curliated or noncurliatedstrains. Incubation of plasma alone served as the control (FIG. 25A).Coincubation with curliated bacteria SR11B and YMel resulted in adramatic increase of bradykinin release (100- and 30-fold, respectively,over control), whereas noncurliated strains YMel-1, CsgA and CsgBliberated only minor amounts of kinins. Collectively, these data showthat curliated bacteria have the capacity to absorb contact-phasefactors from plasma, to activate the contact-phase system and to releasebradykinin from kininogen.

Curliated bacteria prolong the clotting time.

In another series of experiments, we studied the effect of curliatedbacteria on clot formation by measuring the activated partialthromboplastic time (aPTT). Addition of curliated strains YMel and SR11Bto plasma drastically prolonged the clotting time (FIG. 25b), whereasmutants YMel-1, CsgA and CsgB were without effect. The effect of YMel onthe aPTT was dose-dependent (FIG. 25B) as was that of SR11B (data notshown). Physical separation of the bacteria from plasma beforeperforming the apTT assay did not influence the clotting time (data notshown).

Prolonged clot formation is typically caused by a deficiency or defectof coagulation factors. Thus, the plasma samples were incubated withvarious bacterial strains, and following centrifugation theconcentrations of selected coagulation factors were determined in thesupernatants (Table 31). There was no significant consumption of factorX, protein C or antithrombin III. F XI concentrations were slightlyreduced following preincubation with YMel and SR11B but not with YMel-1,CsgA or CsgB. However, the concentrations of fibrinogen weresignificantly lower in plasma preabsorbed with YMel and SR11B, andalmost unaffected in plasma exposed to the mutants. The most pronouncedeffect was seen for the F XII concentration, which dramatically droppedupon incubation with curliated bacteria (Table 3). Hence, curliatedbacteria deplete F XII and other coagulation factors such as fibrinogen,thereby prolonging the clotting time.

TABLE 3 Concentration of coagulation factors in human plasma followingabsorption with different bacterial strains Strain Factor Factor FactorFibrogen Protein C ATT III (%) X* (%) XI* (%) XII* (g/l) (IU/ml) (IU/ml)(IU/ml) YMel 58 38 5 <0.8 0.68 0.56 YMel-1 71 48 48 1.65 0.58 0.56 SR11B64 37 6 <0.8 0.71 0.58 CsgA 58 61 45 1.62 0.6 0.58 CsgB 60 66 44 1.90.56 0.57 Plasma 71 59 58 1.62 0.58 0.54 *The concentration wasexpressed as a percentage of the activity of the factors in poolednormal human plasma

Electron microscopic analysis of clots.

Clots were induced in plasma, in the presence or absence of bacteria,and analyzed by scanning electron microscopy. Clot formation in theabsence of bacteria resulted in long, polymerized fibrin fibrils.Presence of YMel and SR11B bacteria dramatically changed clotmorphology: incorporation of bacteria into the clot resulted in areduced length and diameter of the fibrin fibrils and in a disturbanceof the fibrin network, whereas noncurliated mutants had little effect onthe network morphology. These results suggest that the depletion ofcontact-phase proteins and clotting factors by curliated bacteria andtheir incorporation into the fibrin network disturbs clot formation,prolongs the clotting time, and changes fibril morphology.

To test their effects in animals, the curliated strains YMel and SR11B,as well as the mutant strains YMel-1 and CsgA, were injectedintravenously into mice; vehicle alone served as the control. Bloodsamples were drawn 15 minutes after injection, clots were allowed toform and were analyzed by electron microscopy. In the absence ofbacteria, the clots mainly consisted of erythrocytes that werecrosslinked by an extensive fibrin network. The general organisation ofthe fibrin network, that is the length and diameter of the fibers, wasreminiscent of that formed by normal plasma. In contrast, clots frommice injected with curliated YMel and SR11B were almost devoid of afibrin network, whereas clots from control animals infected withnoncurliated YMel-1 and CsgB had a normal architecture.

Curliated bacteria induce a hypocoagulatory state in vivo

Determinations of the clotting time (aPTT) of plasma samples from miceinfected with the E. coli strains Ymel and YMel-1 were in line withthese observations. Thus, no clots were formed in samples from miceinfected with curliated YMel bacteria even 500 seconds after initiationof the coagulation cascase (FIG. 26A), which corresponded to an almostcomplete depletion of fibrinogen in the samples as measured by asemiquantitative method (FIG. 26B). The clotting time and fibrinogenlevel of plasma samples from mice infected with noncurliated YMel-1bacteria were only slightly affected as compared with those of samplesfrom mice given vehicle alone (FIGS. 26A and B). These in vivo data arefully compatible with the in vitro results and demonstrate thatexperimental infections with curliated bacteria cause blocking of theintrinsic pathway of coagulation and disturbance of fibrinpolymerization through fibrinogen depletion.

Example 6 Activation of the contact phase system by streptococci

Bacteria strains and culture conditions—S. pyogenes strains AP1 (40/58)of the M1 serotype and AP6 (2/66) of the M6 serotype are from the WorldHealth Organisation Collaborating Centre for References and Research onStreptococci, Institute of Hygiene and Epidemionlogy, Prague, CzechRepublic. Streptococci, grown in Todd-Hewitt (Difco) at 37° C. for 16 hwere harvested by centrifugation (2000 g, 20 min), washed in 50 mMHEPES, 2 mM, CaCl₂, 50 μM ZnCl₂, pH 7.35 containing 0.02% (w/v) NaN₃ and0.05% (v/v) Tween 20 (buffer A), and resuspended in buffer A.

Sources of proteins—HK and PK were isolated from plasma, F XI and F XIIwere from Enzyme Research Laboratories, Swansea, UK. Polyclonal antiseraagainst HK were produced in sheep, antisera against PK in rabbits.Antibodies against human F XII were from The Binding Site Limited,Birmingham, UK, and antisera against human F XI were from NordicImmunological Laboratories, Tilburg, The Netherlands.Peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad, Richmond, Calif.),and donkey anti-sheep IgG (ICN, Aurora, Ohio) were used as secondaryantibodies. Proteins were iodinated by the Chloramin-T method of Frakerand Speck, Biochem Biophys. Res. Commun 1978 80 840-857.

Bacterial binding assay—Bacteria were resuspended in buffer A to 2×10⁸cells per ml. 200 μl of the bacterial solution was incubated with theradiolabelled protein (2×10⁴-4×10⁴ cpm) in 25 μl buffer A, at roomtemperature for 10 to 120 min. After incubation and washing, the pelletwas resuspended in 20 μl SDS-PAGE sample buffer containing 1% (v/v)Nonidet P-40 (NP-40) and β-mercaptoethanol. The samples were boiled for5 min followed by centrifugation (3000×g, 5 min) and SDS-PAGE. The fourcontact phase proteins were also added together to the bacteria. Thefollowing concentrations were used: 3.0×10⁻¹² M F XII, 3.9×10⁻¹² M PK,0.2×10⁻¹² M F XI and 4.6×10⁻¹² M HK. In each experiment one of the fourproteins was radiolabelled. Following incubation, bacteria were treatedas above. Radioactivity released from bacteria was analysed byautoradiography. Dried gels were exposed on Kodak Scientific ImagingFilm for one to four days.

SDS-polyacrylamide gel electrophoresis (PAGE) and Westernblotting—Proteins were separated by SDS-PAGE, visualised byautoradiography or transferred onto nitrocellulose membranes for 30 minat 100 mA. The membranes were blocked with 50 mM KH₂PO₄, 0.2 M NaCl, pH7.4, containing 5% (w/v) dry milk powder and 0.05% (v/v) Tween 20.Immunoprinting of the transferred proteins was done. The first antibodywas diluted 1:1000 in the same buffer as described above. Bopundantibody was detected by a peroxidase conjugated secondary antibodyagainst sheep or rabbit IgG identified by the chemiluminescence method.

Absorption experiments—Plasma absorption experiments were performed. Oneml fresh human plasma was incubated with bacteria (2×10¹⁰ bacterialcells/ml) suspended in 1.0 ml buffer A for 60 min at room temperature.After centrifugation (3000×g for 5 min), the cells were washed threetimes with 1.5 ml of buffer A and absorbed proteins were eluted with 0.2ml 30 mM HCl, pH 2.0. The bacteria were centrifugated and thesupernatant was neutralised with 20 μl 1 M Tris, pH 8.0, 0.2 mlSDS-sample buffer was added and the mixture was boiled for 5 min. Elutedproteins were analysed by SDS-PAGE and Western blotting using antibodiesagainst HK, F XII, F XI or PK.

Analysis of proteolytic activity at bacterial surface—0.5 ml plasmaincluding proteinase inhibitors (10 mM Pefabloc, Roth, Karlsruhe,Germany; 10 mM PMSF, Sigma Chem Co, St. Louis, Mo., 10 mM Benzamidine,Sigma Chem, Co, St. Louis, Mo.) were incubated with 2×10¹⁰ cells/ml in0.5 ml buffer A for 10 min at room temperature. The bacteria werecentrifuged and washed three times in buffer A. To measure theproteolytic activities, the bacteria were resuspended in 0.25 ml of a0.6 mM solution or S-2302 (H-D-Pro-Phe-Arg-pNA:2HCl) or S-2366(H-D-Glu-Pro-Arg-pNA:2HCl; Haemochrom Diagnostica, Essen, Germany) in0.15 M Tris-HCl, pH 8.3. After 30 min the bacteria were centifuged andsubstrate hydrolysis in the supernatant was measured at 405 nm.

Clotting assay—The clotting time was monitored in an Amelungcoagulometer (Amelung, Lemgo, Germany). 30 μl of a solution of 2×10¹⁰bacteria resolved in 100 μl of a 50 mM HEPES, 50 μM ZnCl₂, pH 7.35solution were incubated with sodium citrate-treated plasma for 30 s,followed by incubation with Platelin® LS aPTT (Organon Teknica, Durham,N.C.) or with phospholipids alone (37.5% phosphatidyl choline, 37.5%phospatidylethanolamine and 25% phosphatidyl serine; all from SigmaChem. Co, St. Louis, Mo.) for 200 s. Coagulation was initiated by adding100 μl of a 25 mM CaCl₂ solution.

Scanning electron microscopy—For scanning electron microscopy (SEM), theclots were gently drawn down onto a wet Millipore filter (WatersCorporation, Milford, Mass.) by suction caused by presetted filter paperlying underneath. Subsequently the whole filter was fixed in 2% (v/v)glutaraldehyde in 0.1 M sodium cacodylate plus 0.1 M sucrose, pH 7.2,for 1 h at 4° C. and the washed with 0.15 M cacodylate buffer, pH 7.2.The filters were then washed with cacodylate buffer, and postfixed for 1h in 1% (w/v) osmium tetroxide in 0.15 M sodium cacodylate, pH 7.2 for 1h at 4° C., washed, and stored in cacodylate buffer. Fixed filter papersample were dehydrated for 10 min at each step of an ascending ethanolseries, critical point dried, mounted on aluminium holders,palladium/gold sputtered, and examined in a Jeol JSM-350 SEM.

S. pyogenes of strain AP1 was grown as described above. The bacteriawere washed twice with sterile PBS, and resuspended in sterile PBS to aconcentration of 2×10¹⁰ cells/ml in the absence or presence of 0.2 mg/mlof F XII inhibitor H-D-Pro-Phe-Arg-CMK (Bachem, Switzerland). Femaleoutbred NMRI mice were given an intravenous injection of 200 μl vehicleor 200 μl bacterial suspension, during ether anaesthesia. 15 minfollowing the injection, the animals were again anaesthetized and bloodsamples were taken by punction of the heart. Animals were killed bycervical dislocation.

Assembly of contact phase proteins on streptococcal surfacestructures—In order to study the influence of streptococci on theintrinsic pathway of coagulation, binding of all contact phase proteinsto the streptococcal serotypes AP1 and AP6 was investigated. In directbinding assays we found, that radio-labelled contact phase factors areabsorbed on the surfaces on the strains AP1 and AP6 (data not shown),whereas the Gram-negative strain CsgA which does not express curli fromSalmonella typhimurium failed to show a significant binding. Weincubated the radio-labelled proteins with AP1 and AP6 bacteria andanalysed absorbed proteins by SDS-PAGE. Only F XII was degradedresulting in two fragments of 50 kDa and 30 kDa, respectively. In thiscontext it is noteworthy to mention, that limited proteolysis is thegeneral mechanism of converting coagulation factors to their activeforms. Since the F XII fragments on the streptococcal surfacescorrespond to heavy and light chain of the active enzyme, the resultsindicate that the molecule is activated upon binding to streptococci.However, no degradation of HK, PK and F XI was detected and F XII wasnot cleaved when incubated in the absence of bacteria under the sameconditions (data not shown).

The interaction between F XII and the other members of the contact phasesystem was analysed by incubating bacteria with all four proteinstogether, whereby the molar ratios of the coagulation factors were thesame as in plasma and only one protein was radioactively labelled.Radiolabelled PK was only cleaved in the presence of F XII, HK and F XIwhen incubated with bacteria. No degradation of PK was monitored in theabsence of these factors. In analogue experiments, radio-labelled HK wasalso only cleaved in the presence of all four factors when applied tothe bacteria (FIG. 27). The resulting HK fragments of 65 kDa and 55 kDacorrespond to heavy and light chain of kinin-free HK. Under identicalconditions but in the absence of bacteria no degradation ofradio-labelled contact phase factors was observed (data not shown).Taken together these results indicate that the binding of contact phasefactors to the streptococcal surfaces initiate the contact phase systemvia initial activation of F XII.

Contact phase proteins are captured from plasma by streptococci—Inanother series of experiments we investigated the interaction betweenbacteria and contact phase proteins under physiological conditions, inthat the streptococcal strains AP1 and AP6 were incubated with humanplasma. After extensive washing, absorbed proteins were eluted from thebacterial surface and run on SDS-PAGE followed by transfer onnitrocellulose. Separated proteins were probed by antibodies against FXII, HK, PK and F XI. As a control, the non-binding strain CsgA wasused. AP1 and AP6 captured all four contact phase proteins from plasmawhereas CsgA failed to show significant binding. Interestingly, theproteins were degraded. These finding support the notion that thecontact phase system is also triggered under physiological conditions.

The amidolytic activity of the streptococcal absorbed factors wasstudied in chromogenic assays. The proteolytic assay was establishedsuch that bacteria were incubated with plasma and, after an extensivewashing procedure, they were resuspended in a buffer containing thespecific substrates for F XI, H-D-Glu-Pro-Arg-pNA (S-2366), and for FXII/PK, H-D-Pro-Phe-Arg-pNA (S-2302). To this end proteolytic activitiesof the F XI and F XII/PK were found on the surfaces of AP1 and AP6,whereas the activities observed on the surface of control strain CsgAwere significantly reduced (FIG. 28). When we tested the endogenousactivities on bacterial surfaces, none of these strains was able tohydrolyse any of the substrates in the absence of plasma (data notshown). The findings described above are in accordance with our previousreported data showing that streptococcal bound HK releases bradykinin byPK treatment. Thus, the experiments demonstrated, that afterstreptococcal contact with plasma the bacteria are able to assembly allfour coagulation factors followed by activation of the contact phasesystem.

Influence of streptococci on the clotting time—To test the influence ofstreptococci on clot-formation we employed the kaolin activated partialthromboplastin time (aPTT) assay. In these experiments, plasma waspre-incubated with bacteria followed by measurements of the resultingaPTT values. As shown in FIG. 29A, AP1 and AP6 significantly prolongedthe clot-formation as compared to plasma in absence of bacteria. Thecontrol strain CsgA had only a small effect on the APTT. Similar resultswere obtained for the clotting time in the absence of kaolin (FIG. 29B),These results were unexpected wince the activation of the contact phasesystem should initiate the coagulation system. However, prolongation ofclot formation occurs normally by deficiency of coagulation factors inplasma.

We tested if a consumption of coagulation factors by the bacteria wasresponsible for the observed effect. The experiments were constructedsuch that plasma and bacteria were incubated for 30 min followed byremoving bacteria from plasma by centrifugation. The resultingsupernatants were used for the determination of the kaolin activatedaPTT. As observed before, pre-incubation of plasma with AP1 and AP6prolonged the clot-formation whereas the CsgA failed to induce a delayin the same set-up (FIG. 29C). These data suggest, that not the presenceof bacteria but a consumption of coagulation factors induced thiseffect. Analysis of the plasma-supernatants after incubation withbacteria revealed, that fibrinogen was almost completely depleted whenplasma was pre-incubated with AP1 and AP6. No fibrinogen depletion wasobserved in samples treated with CsgA in the same experiment (data notshown). Finally, these measurements demonstrated that, despite theinitiation of the contact phase system on streptococcal surfaces,consumption of fibrinogen and probably other coagulation factors induceda disturbance in clot formation.

Electron microscopic studies of clots formed from plasma in the presenceof bacteria. In further studies morphological alternations of clots inpresence of streptococci were investigated. Clots formed from humanplasma in the absence of kaolin with and without bacteria were analysedby electron microscopy. As shown in figure, control clots withoutbacteria are built by long, thick fibrin polymers forming a fibrinnetwork. In presence of AP1 and AP6 no fibrin network formation wasdetectable and bacteria were inserted into the clots. In contrast, clotsformed in the presence of CsgA bacteria showed a normal fibrin networkformation as observed in the control clot and no bacteria were insertedinto the clot. Taken together these results imply that the ability ofstreptococci to bind to and to activate contact phase proteins on theirsurfaces results in a prolongation of the clotting time, a change infibrin fibril morphology and an incorporation of bacteria inside theclots.

To study the interaction between streptococci and the contact phasesystem in vivo an animal model was established. In these experiments,mice were intravenously injected with AP1 and AP6 and alternatively withthe same bacteria in the presence of the F XII inhibitorH-D-Pro-Phe-Arg-CMK. As a control vehicle along was applied. A shorttime after injection blood samples were taken from the animals and clotswere formed, which were in addition analysed by electron microscopy.Clots created in the absence of streptococci were built by arythrocytescross linked via a fibrin network. However, when AP1 and AP6 wereinjected into the animals clots were formed by densely packederythrocytes in the absence of a fibrin network and bacteria wereincorporated in the clots. When bacteria were injected together with theF XII inhibitor, clots appeared as observed in the control resulting inprecipitated erythrocytes cross linked via a fibrin network and nobacteria were incorporated in the clots. From the electron-micrographsit can be concluded that the presence of streptococci in the bloodstreaminduces dysfunctions in clot-formation and that inhibition of F XIIactivity abolished this effect. Thus, in conclusion, these datademonstrated that the activation of the contact phase system duringstreptococcal infection is combined with a change in the coagulationproperties of the host.

What is claimed is:
 1. An assay for compounds useful in the treatment ofbacterial induced coagulation disorders, comprising: (a) incubating aplasma sample with a strain of bacteria; (b) adding a compound to beassayed to the plasma sample before, during or after step (a); (c)conducting an activated partial thromboplastin time test after steps (a)and (b); and (d) determining the clotting time; wherein a decrease inclotting time indicates a compound useful in the treatment of bacterialinduced coagulation disorders.
 2. The assay according to claim 1 inwhich the bacteria are removed from the sample prior to step (b) or step(c).
 3. An assay for compounds useful in the treatment of bacterialinduced coagulation or inflammatory disorders, comprising: (a)incubating a sample of bacteria with one or more contact phase proteinsselected from the group consisting of H-kininogen, prekallikrein, factorXII and factor XI; (b) adding a compound to be assayed before, during orafter step (a); and (c) determining the binding of the contact phaseproteins to the bacterial surface; wherein a decrease in the binding ofsaid contact phase proteins to said bacterial surface indicates acompound useful in the treatment of bacterial induced coagulation and/orinflammatory disorders.
 4. An assay for compounds useful in thetreatment of bacterial induced coagulation or inflammatory disorders,comprising: (a) incubating a strain of bacteria with one or more contactphase proteins selected from the group consisting of H-kininogen,prekallikrein, factor XII and factor XI; (b) adding the compound to beassayed before, during or after step (a); and (c) determining theactivation of the or each contact phase protein; wherein a decrease inthe activation of the contact phase protein or each contact phaseprotein indicates that said compound is useful in the treatment of abacterial induced coagulation and/or inflammatory disorder.
 5. The assayaccording to claim 4 wherein H-kininogen is present in the sample andactivation is monitored by release of bradykinin.